Effect of the superoxide radical on lymphocyte proliferation stimulated by a mitogen

Administration of superoxide dismutase or a complex of divalent copper with lysine to human and mice lymphocyte cultures was shown to prevent from the lymphocyte proliferative response to concanavalin A stimulation. The inhibition grade dependence on the inhibitor concentration in culture medium was studied. It is concluded that active oxygen forms produced by macrophages may be factors defining the value of proliferative response of lymphoid cells under mitogen stimulation.     

Inhibition of mitogen and specific antigen-induced human lymphocyte proliferation by cadmium

Cadmium chloride (CdCl2) added to human lymphocyte culture inhibits the proliferative response induced by phytohaemagglutinin (PHA), pokeweed mitogen and allogenic lymphocytes in mixed lymphocyte reaction. Minimally effective concentrations of CdCl2 were 3.3, 1.6 and 1.6 microM, respectively. The inhibition was greatest when CdCl2 was added at initiation of cultures and declined if the addition of CdCl2 was postponed. The presence of CdCl2, regardless of the presence of PHA during the first 24 h of incubation suppressed the proliferative response to subsequent stimulation with PHA, indicating that cadmium affects an early step of blastogenic transformation.     

Human placental Hofbauer cells promote mitogen induced T cell proliferation

Hofbauer cells are a major cell type of the human placental villous core particularly numerous at the beginning of pregnancy. In the present study we have investigated whether or not Hofbauer cells could subserve the function of accessory cells for colony formation by phytohemagglutinin stimulated allogeneic T cells. Results showed that Hofbauer cells are capable to play an accessory role on T cell proliferation probably due to the release of interleukin 1-like soluble factors.     

Inhibition of the mixed lymphocyte reaction by fractionated niridazole urine dialysate.

Urine dialysate from rats treated orally with 25 mg/Kg 3H-labeled niridazole was fractionated by DEAE-Sepharose column chromatography and was found to contain three radioactive metabolites and no parent compound. When human niridazole urine dialysate (NUD) was fractionated under identical conditions, fractions corresponding to the three rat NUD metabolites were found to inhibit the human one-way MLR. No inhibition was obtained with fractionated control urine dialysate. It was concluded that nonimmunosuppressive niridazole is metabolized by rats and man to produce three active compounds with the ability to suppress the in vitro response to alloantigens.     

Mastocytoma-medicated suppression of mixed lymphocyte culture and mitogen responsiveness.

Murine spleen cells, stimulated in vitro by allogeneic spleen, display a strong proliferative response with the subsequent development of cytotoxic cells. This proliferation and sensitization can be abrogated by the addition of mitomycin-treated or X-irradiated murine DBA/2 mastocytoma cells (P-815). The substance required for this depression of lymphocyte responsiveness is present in the cell-free supernatant fluids of P-815 cultures. The suppression appears to be due to interference with cell proliferation in the mixed lymphocyte culture, because the P-815 also prevents spleen cells from proliferating in response to the mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and phytohemagglutinin (PHA). The significance of these findings is discussed.     

Effect of mitogen concentration on glucocorticoid suppression of normal and cystic fibrosis lymphocyte activation

It has previously been demonstrated that glucocorticoid suppression of mitogen-induced lymphocyte activation is a function of mitogen dose. Glucocorticoids suppress lymphocyte activation more at low doses, which induce suboptimal lymphocyte activation, than at higher doses which are optimal for lymphocyte activation. This observation suggests that glucocorticoid suppression of lymphocyte activation might be greater than normal in disease states which are associated with depressed mitogen-induced lymphocyte activation. To test this hypothesis, lymphocytes from normal individuals and patients with cystic fibrosis were activated by a full range of concentrations of concanavalin A (Con A) in the presence or absence of dexamethasone. Con A activation of cystic fibrosis lymphocytes was markedly depressed compared to the activation of normal lymphocytes at all doses of Con A, but the suppressive effect of dexamethasone on the activation of normal and cystic fibrosis lymphocytes was the same. We conclude that glucocorticoid suppression of lymphocyte activation is more a function of mitogen dose than of the level of lymphocyte activation and is not necessarily greater than normal in disease states which are associated with depressed mitogen-induced lymphocyte activation.     

Effect of dietary fat on lymphocyte proliferation and metabolism

The effect of diets enriched with fat containing different fatty acids on glucose and glutamine metabolism of mesenteric lymph nodes lymphocytes, spleen, and thymus and lymphocyte proliferation was examined. The following fat-rich diets were tested: (1) standard chow (CC); (2) medium chain saturated fatty acids (MS)—coconut fat oil; (3) long chain saturated fatty acids (LS)—cocoa butter; (4) monounsaturated fatty acids (MU)—canola oil (n-9); (5) polyunsaturated fatty acids (PU)—soybean oil (n-6). Of the fat-rich diets tested, MS was the one to present the least pronounced effect. Lymphocyte proliferation was reduced by LS (64 per cent), MU (55 per cent), and PU (60 per cent). Hexokinase activity was enhanced in lymph node lymphocytes by PU (67 per cent), in the spleen by MS (42 per cent), and in the thymus by PU (30 per cent). This enzyme activity was reduced in the spleen (33 per cent) by LS and MU (35 per cent). In the thymus, this enzyme activity was reduced by LS (26 per cent) and MU (13 per cent). Maximal phosphate-dependent glutaminase activity was raised in lymphocytes by MS (70 per cent) and MU (20 per cent). This enzyme activity, however, was decreased in lymphocytes by PU (26 per cent), in the spleen by LS (15 per cent), and in the thymus by MU (44 per cent). Citrate synthase activity was increased in lymphocytes by MU (35 per cent), in the spleen by LS (56 per cent) and MU (68 per cent), and in the thymus by LS (42 per cent). This enzyme activity was decreased in lymphocytes by PU (24 per cent) only. [U-¹⁴C]-Glucose decarboxylation was raised by all fat-rich diets; MS (88 per cent), LS (39 per cent), MU (33 per cent), and PU (50 per cent), whereas [U-¹⁴C]-glutamine decarboxylation was increased by LS (53 per cent) and MU (55 per cent) and decreased by MS (17 per cent). The results presented indicate that the reduction in lymphocyte proliferation due to LS, LU and PU could well be a consequence of changes in glucose and glutamine metabolism. Copyright © 1998 John Wiley & Sons, Ltd.     

Differential inhibition of mitogen induced T cell proliferation by 5-azacytidine and cytosine-arabinoside

The cytotoxic drugs 5-azacytidine and cytosine-arabinoside influence the enzymatic methylation of DNA in opposite ways (1,2). The in vitro effects of these two drugs on Con A induced proliferation of thymic and splenic rat lymphocytes were investigated. Cytosine-arabinoside was found to inhibit mitogen induced proliferation already at a concentration of 0.001 microM, whereas 5-azacytidine was inhibitory only above concentrations of 1 microM. A stimulation of mitogen induced T cell proliferation was consistently seen with 5-azacytidine, but not with cytosine-arabinoside, at concentrations lower than the cytotoxic concentration. The results show that 5-azacytidine and cytosine-arabinoside interfere with mitogen stimulated lymphocyte proliferation by different mechanisms and suggest that hypomethylated DNA plays a role in the proliferation of T cells.     

Maintenance of lymphocyte surface Ig by mitogen stimulation in vitro.

In 4 to 24 hr cultures of rabbit lymphoid cells in medium supplemented with autologous serum, most B cells lost their surface Ig as assayed by rosette formation with anti-Ig antibody-coated erythrocytes. This loss was prevented by adding selected mitogens such as streptococcal mitogen (SM), lipopolysaccharide, and concanavalin A or by supplementing the medium with fetal calf serum. When SM was added at various times to the cultures (1, 2, 3, and 4 hr), it was effective in maintaining the approximate level of Ig-bearing cells present at the time of its addition but was ineffective in restoring the level of Ig-bearing cells present at the time the cultures were intiated. Very small, submitogenic doses of SM were sufficient to maintain the level of Ig-bearing cells. The data suggest that lymphocytes require continuous stimulation to maintain their surface receptors.     

Plasminogen activator is an apparent lymphocyte mitogen

Culture fluids of avian sarcoma virus (ASV)-transformed but not normal chicken embryo cells frequently elicited a mitogenic response in normal avian and murine lymphocytes. We examined the possibility that plasminogen activator (PA) might be responsible for the observed mitogenic effect. PA activity, present in culture medium, was correlated positively with lymphocyte mitogenic capacity. Treatment of cells with phorbol myristate acetate, which elevates PA levels, increased mitogenesis. Similar treatment with dexamethasone, which inhibits PA biosynthesis and/or secretion, reduced lymphocyte mitogenic activity. Addition to culture fluids of either benzamidine or diisopropylfluorophosphate, both specific PA inhibitors, blocked lymphocyte proliferative responsiveness to culture fluids. In contrast, neither epsilon-amino-caproic acid nor trasylol, which inhibits plasmin esterase activity but not PA, abrogated lymphocyte responsiveness. Furthermore, purified urokinase, an enzyme of similar substrate specificity to PA, had lymphocyte stimulatory activity. These results strongly suggest that PA can function as a lymphocyte mitogen.     

Effect of exogenous calmodulin on lymphocyte proliferation in healthy individuals

The action of calmodulin (CM) given in doses of 10(-5)-10(-8) M on lymphocyte proliferation in healthy subjects was studied. All the test doses of CM produced a stimulant effect 72 h after the lymphocyte incubation was commenced. The rate of 3H-thymidine incorporation experimentally was 1.5-3 times higher than in the control. It is assumed that the release of CM in the foci of tissue injury or inflammation may serve one of the factors stimulating lymphocyte proliferation in an individual focus.     

Modulation of lymphocyte mitogen responses by cocultured fibroblasts

Immunological reactions in vivo occur in an environment rich in fibroblasts (FB) and other connective tissue cells. The possibility that FB might affect mononuclear cell proliferative responses to mitogens in vitro was examined in a microculture system. Human peripheral blood mononuclear cells (MC) were cocultured with mitomycin C-treated FB in the presence of phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Cocultured FB (at a 1:100 to 1:10 FB:MC ratio) suppressed the response of MC to PHA (by as much as 35%) but did not significantly affect PWM responses. Cocultures of FB and MC were characterized by 10- to 30-fold increases in prostaglandin E₂ concentrations compared to MC or FB cultured alone. Inhibition of prostaglandin synthesis with indomethacin or mefenamic acid significantly reversed the FB-mediated suppression of lymphocyte PHA responses. Prostaglandin-dependent FB suppression of lymphocyte PHA responses was seen only when FB:MC coculture was initiated at the onset of exposure of MC to PHA. When FB were added to MC after 24 hr of culture with PHA, no effect was seen. Addition at 48 or 66 hr resulted in prostaglandin-independent enhancement of lymphocyte proliterative responses to PHA. Thus in cocultures of FB and MC, MC reactivity to PHA may be influenced in part via alterations in FB prostaglandin metabolism. The interaction between FB and MC may be important in the modulation of immune responses in inflammatory lesions.     

Effect of sodium diethyl dithiocarbamate on mitogen induced lymphoproliferation "in vitro"

Sodium diethyl dithiocarbamate (DTC) is a low molecular weight sulphur compound which has been shown to be an adjuvant of immunity in vivo. In this report, DTC was shown to inhibit in vitro DNA-synthesis of human peripheral blood lymphocytes stimulated by phytohemagglutinin or concanavalin A. DTC could act directly at the cellular level and indirectly by interfering with culture medium or lectins.     

Effect of halofantrine and its desbutyl metabolite on lymphocyte proliferation dynamics

Halofantrine hydrochloride (HF), one of the latest antimalarial agents currently undergoing clinical trials, and its active metabolite, N-desbutylhalofantrine (DHF), were examined for their effects on human and rat lymphocytes. HF has a biphasic concentration-dependent effect on phytohemagglutinin stimulated proliferation of human lymphocytes. Concentrations lower than 2.25 μM enhance, while higher concentrations inhibit proliferation. The IC₅₀ values were 9.4 μM for HF, 4.5 μM for DHF and 14.7 μM for chloroquine. In human lymphocytes, enhanced proliferation was not detected for DHF unlike for HF. Combined achievable plasma concentrations of HF and DHF may sometimes be in the range where reduced lymphocyte proliferation occurs in vitro when based on simple additive dynamics. It remains to be confirmed if malarial treatment with HF leads to reduced T-cell responsiveness to antigenic challenges since HF and DHF persist for several days.     

Sequential proliferation induced in human peripheral blood lymphocytes by mitogen. I. Growth of 1000 lymphocytes in feeder layer cultures.

A culture system is described in which 1000 human peripheral blood lymphocytes diluted in 2.5 x 10(5) mitomycin-treated autologous cells respond to phytohemagglutinin (PHA). Proliferation data, including 3HTdR uptake, cell survival counts, and mitotic indices, indicate that this inoculum expands from 1000 to 40,000 cells by day 7, suggesting five or six sequential cell divisions. Chromosome markers in allogeneic cultures demonstrate that the dividing cells are derived from the original 1000 cells and not from the "feeder layer" of mitomycin-treated lymphocytes. The time course of proliferation in this system is similar to that in other human lymphocyte culture systems with a low percentage of responding cells, as in the response to PHA of cells from patients with chronic lymphocytic leukemia or the response of normal lymphocytes to antigens. The conditions provided by the feeder layer which permit proliferation of this small number of lymphocytes are not precisely known, but erythrocytes, heat-killed lymphocytes, or inert particles do not provide a satisfactory substitute.     

Enhancement of intracellular glutathione promotes lymphocyte activation by mitogen

We have examined the effect of chemically modulating intracellular glutathione (GSH) levels on murine lymphocyte activation. Lymphocyte activation was determined by the induction of polyamine synthesis (ornithine decarboxylase (ODC) induction) and DNA synthesis ([3H]thymidine([3H]Tdr) incorporation). Intracellular GSH levels were enhanced using L-2-oxothiazolidine-4-carboxylate (OTC), which delivers cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO), which inhibits gamma-glutamylcysteine synthetase. In addition, the thiol 2-mercaptoethanol (2-ME) was tested for its ability to augment intracellular GSH levels. Our results indicate that both OTC and 2-ME enhance GSH concentrations and [3H]Tdr incorporation in resting and mitogen (concanavalin A)-stimulated cells. The induction of ODC by concanavalin A (Con A) was augmented by the addition of OTC or 2-ME. The GSH concentration of Con A-stimulated cells was reduced when compared to resting cells; however, it was markedly enhanced by OTC or 2-ME. The stimulatory effects of 2-ME on GSH concentrations, [3H]Tdr incorporation, and ODC induction in both resting and Con A-stimulated cells were much more potent than those of OTC. In contrast, BSO suppressed intracellular GSH and [3H]Tdr incorporation in resting and Con A-stimulated cells. BSO also inhibited the promotion of intracellular GSH concentrations and [3H]Tdr uptake by OTC or 2-ME. However, BSO did not affect the induction of ODC by Con A or its enhancement by OTC or 2-ME. We conclude that enhancement of intracellular GSH concentration results in an increased lymphocyte response to mitogen stimulation.     

The requirement for macrophage-lymphocyte interaction in T lymphocyte proliferation induced by generation of aldehydes on cell membranes.

Guinea pig T lymphocyte proliferation induced by sodium periodate (NaIO4) or neuraminidase-galactose oxidase (NG) occurs when lymphocytes and macrophages are cultured together after treatment of either purified T lymphocytes or macrophages with these agents. Regardless of which cell initially bears the modified surface carbohydrate, lymphocyte proliferation requires the presence of viable homologous macrophages and fails to occur when they are replaced with fibroblasts, erythrocytes, L2C leukemia cells, thymocytes, PMN, line I hepatoma cells, or murine macrophages. Lymphocyte proliferation resulting from NaIO4 or NG treatment of lymphocytes is diminished when these cells are treated with proteolytic enzymes or aged in in vitro culture for 48 hr. By contrast, proteolytic enzyme treatment or in vitro aging has no effect on the ability of NaIO4 or NG-treated macrophages to induce lymphocyte proliferation. The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophage in the initiation of T lymphocyte proliferation.     

Modulation of lymphocyte proliferation induced by gastric MALT lymphoma-associated Helicobacter pylori strains

Background: Helicobacter pylori infection leads to different chronic diseases, suggesting that this bacterium can evade the host immune defense system. The ability to control lymphocyte proliferation may be a mechanism leading to the development of gastric pathologies. Our aim was to characterize the effects of mucosa‐associated lymphoid tissue (MALT) associated H. pylori strains on lymphocyte proliferation. Materials and methods: We measured the in vitro proliferation of human lymphocytes originally from blood or tonsil samples in the presence or absence of viable bacteria or lysates. Results: We showed that MALT lymphoma‐associated strains are not likely to be directly responsible for anarchical B‐cell proliferation in vitro. On the other hand, proliferation of prestimulated T lymphocytes was abolished in vitro by the presence of all H. pylori strains, whether associated with MALT lymphoma or not. Conclusion: Inhibition of T‐cell proliferation may be of major importance in the gastric colonization and in the persistence of the infection. Furthermore, this inhibition may favor anarchical B‐cell proliferation in vivo and predispose the host to gastric MALT lymphoma, whereas MALT‐associated H. pylori strains do not appear to possess a specific capability to directly stimulate B‐lymphocyte proliferation.