Unsaturated fatty acids suppress interleukin-2 production and transferrin receptor expression by concanavalin A-stimulated rat Iymphocytes

The proliferation of T-lymphocytes is dependent upon their ability to synthesize and secrete the cytokine, interleukin-2, and to express cell surface receptors for interleukin-2 and transferrin. We have previously reported that certain fatty acids inhibit mitogen-stimulated T-lymphocyte proliferation. We now report that unsaturated fatty acids decrease the concentration of interleukin-2 in the culture medium of such cells by up to 45%. This suggests that unsaturated fatty acids inhibit lymphocyte proliferation by suppressing interleukin-2 production. However, lymphocyte proliferation was only partially restored by addition of exogenous interleukin-2 to cell culture medium in the presence of unsaturated fatty acids, indicating that these fatty acids also affect other processes involved in the control of proliferation. Saturated fatty acids, which also inhibit lymphocyte proliferation, did not affect the interleukin-2 concentration in the culture medium suggesting a different mechanism for their action. Neither saturated nor unsaturated fatty acids affected the expression of the interleukin-2 receptor by mitogenstimulated lymphocytes. In contrast, unsaturated fatty acids decreased expression of the transferrin receptor by up to 50%. These observations suggest that the mechanism by which unsaturated fatty acids inhibit lymphocyte proliferation involves suppression of interleukin-2 production and of transferrin receptor expression. The mechanism for the inhibitory action of saturated fatty acids is not clear.     

DNA topoisomerase activities in concanavalin A-stimulated lymphocytes

Topoisomerase activities have been measured in nuclear extracts of concanavalin A-stimulated lymphocytes. In parallel with the wave of DNA synthesis, type II topoisomerase activity was considerably increased. After 72 h treatment, this activity was stimulated approx. 20-fold over the activity in untreated cells. In contrast, type I topoisomerase was poorly stimulated after 24 h treatment, and 4-5-fold after 72 h. These findings, together with our previous results on regenerating rat liver, suggest a major role of topoisomerase II in DNA replication.     

Effect of macrophages on the translocation of protein kinase C in concanavalin A-stimulated lymphocytes

The concanavalin A (Con A)-induced proliferation of lymph node lymphocytes is dependent on the presence of macrophages. When lymphocytes are depleted of macrophages, Con A is no longer mitogenic. Either 12-0-tetradecanoylphorbol-13-acetate (TPA), interleukin 1 (IL1), or macrophages in combination with Con A can restore proliferation. To establish where the proliferation process is blocked in the absence of macrophages, an early step in the signalling pathway, the activation of protein kinase C, was examined. It was found that although Con A caused translocation of protein kinase C from the cytosol to the membrane of lymph node cells, when the lymph node cells were depleted of macrophages and exposed to Con A, this translocation of protein kinase C did not occur. Instead, protein kinase C activity decreased in the membrane fraction and increased in the cytosol. On the other hand, TPA caused translocation of protein kinase C (PKC) from the cytosol to the membrane regardless of the presence of macrophages. However, the macrophage product, IL1, alone or in combination with Con A did not cause translocation of protein kinase C. In a reconstitution experiment, in which lymph node cells were depleted of macrophages and then macrophages were added back, the addition of Con A again lead to translocation of protein kinase C from the cytosol to the membrane. This combination also restored cell proliferation. Therefore, the Con A induced PKC translocation in T lymphocytes is macrophage mediated. TPA overcomes the macrophage requirement by directly activating PKC, while IL1 appears to act at a different step in proliferation.     

Arachidonic acid-containing phosphatidylcholine inhibits lymphocyte proliferation and decreases interleukin-2 and interferon-gamma production from concanavalin A-stimulated rat lymphocytes

The proliferation of concanavalin A (Con A)-stimulated rat lymphocytes was markedly inhibited by phosphatidylcholine containing arachidonic and stearic acids (PC(A-S)), but not by phosphatidylcholine containing oleic and stearic acids or phosphatidylinositol containing arachidonic and stearic acids. The concentration of PC(A-S) which inhibited Con A-stimulated proliferation by 50% was 31 microM and near total inhibition was observed at 154 microM . Phosphatidylserine containing only oleic acid enhanced proliferation by 37% at a concentration of 31 microM , but phosphatidylethanolamine and phosphatidylcholine containing only oleic acid did not affect proliferation at this concentration. It is concluded that both the head group and the fatty acid composition contribute to the influence of phospholipids on lymphocyte proliferation. The effects of PC(A-S) on T-lymphocyte responses were investigated further. In parallel with the inhibition of proliferation PC(A-S) caused a concentration-dependent decrease in the production of the Th1-type cytokines interleukin (IL)-2 and interferon (IFN)-gamma; inhibition of cytokine production was >85% at the highest concentration of PC(A-S) used (154 microM ). Production of the Th2-type cytokines IL-4 and IL-10 was not affected. The possible role of prostaglandins in mediating the effects of PC(A-S) was examined by adding indomethacin into the medium and the participation of lipid peroxidation was examined by adding vitamin E and vitamin C. Indomethacin and vitamin E did not affect the inhibition caused by PC(A-S) but vitamin C caused a partial reversal. It is concluded that inhibition of T-lymphocyte proliferation by phospholipids involves both the head group and the fatty acyl chains, that this inhibition is not mediated by prostaglandins but may involve some form of oxidant stress and that some phospholipids (e.g., PC(A-S)) can markedly influence cytokine profiles.     

Cytochrome changes in control and concanavalin A-stimulated lymphocytes

α-Aminoisobutyrate (AIB) serves as a transportable, nonmetabolizable alanine analog in the purple sulfur bacterium Chromatium vinosum. AIB transport in C. vinosum appears to be catalyzed by an electrogenic Na⁺-alanine (AIB) symport without any direct participation of ATP-driven or H⁺-symport systems. In addition to Na⁺ being cotransported with AIB via the symport, a transmembrane Na⁺ gradient appears to increase the affinity of the symport of AIB. It appears that these two effects of Na⁺ involve different Na⁺-binding sites.     

The effect of colchicine on cholesterol biosynthesis in concanavalin A-stimulated lymphocytes

Attempting to test the hypothesis that colchicine blocks lymphocyte activation at the commitment point, we determined whether or not colchicine inhibited the ConA-induced increase in the synthesis and content of cholesterol in lymphocyte cultures. Colchicine decreased the incorporation of (¹⁴C) acetate into cholesterol by about 50%. However, the decrease affected unstimulated and stimulated lymphocytes equally. The presence of colchicine for as long as 48 hours did not prevent stimulated lymphocytes from accumulating cholesterol. The results suggest that an intact microtubular system is not required for the initiation of blastogenesis and the activation of cholesterol synthesis.     

Nuclear matrix from resting and concanavalin A-stimulated human lymphocytes

Concanavalin A-induced transformation and proliferation of human peripheral blood lymphocytes was found to be accompanied by morphological and biochemical changes of the nuclear matrix. Nuclear matrix spheres increased in size as well as in protein and RNA content. Experimental data suggesting the involvement of the nuclear matrix in DNA replication processes are also presented.     

Effects of Cd2+ upon Ca2+ fluxes and proliferation in concanavalin A-stimulated lymphocytes

The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) is inhibited by micromolar concentrations of CdCl2. This inhibition is partially relieved by an increase in the external Ca2+ concentration (from 0.6 to 2.2 mM). The initial rate of conA-induced 45Ca2+ influx is unaltered by CdCl2, although the level of 45Ca2+ accumulation increases. The basal rate of 45Ca2+ entry is not measurably disturbed by CdCl2 (100 microM). The steady-state efflux of 45Ca2+ and the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of erythrocyte ghosts are inhibited by CdCl2 (10 microM). Thus, the mechanism behind the Cd2+-induced suppression of the mitogenic response to conA is not due to alteration of mitogen-stimulated Ca2+ influx. We suggest that Cd2+ competes with Ca2+ for intracellular Ca2+-binding molecules, such as calmodulin, essential for the induction of cell proliferation.     

Effects of bile acids and lectins on immunoglobulin production in rat mesenteric lymph node lymphocytes

Summary We investigated the effect of bile acids either alone or in combination with lectins on immunoglobulin (Ig) production in vitro of rat mesenteric lymph node (MLN) lymphocytes to examine their immunoregulatory activities. Among free bile acids examined, chenodeoxycholic acid stimulated IgE production by MLN lymphocytes and inhibited IgA production at the concentration of 0.3 mM, whereas cholic and deoxycholic acids exerted the comparable effect at 3 mM. Among conjugated bile acids, deoxycholic acid derivatives stimulated IgE production more strongly than cholic acid derivatives. On the other hand, free and conjugated bile acids did not affect IgG production. The IgE production by MLN lymphocytes was stimulated by concanavalin A and inhibited by pokeweed mitogen, and the effect of phytohemmagglutinin and lipopolysaccharide was marginal. These lectins did not affect IgA and IgG production by the lymphocytes. In the presence of lectins, free bile acids affected IgE production at 0.03 mM. These results suggest the possibility that bile acid is a stimulant for food allergy.     

Relationship between phosphatidylcholine biosynthesis and cellular commitment in concanavalin A-stimulated lymphocytes

The incorporation of [¹⁴C]choline into phosphatidylcholine was studied in lymphocyte cultures exposed to concanavalin A (ConA). The lectin was found to induce an increase in the incorporation of the label with following features:

1. 1. It occurs very promptly after exposure.

2. 2. It is not elicited by a non-mitogenic lectin.

3. 3. The increase in the early stage is proportional to lectin concentration.

4. 4. It can be terminated by a competitive inhibitor of ConA binding.

5. 5. The extent of the increase shows a correlation with the rate of cellular commitment to initiate DNA synthesis. These results suggest that in the mitogenic stimulation of T lymphocytes enhanced synthesis of membrane phospholipids is a precommitment event.

     

Characterization of glutamine transport into resting and concanavalin A-stimulated peripheral human lymphocytes

Characteristics of glutamine transport, its substrate specificity, and its pattern of competitive and non-competitive inhibition in response to amino acid analogues were determined in peripheral human lymphocytes, incubated with or without concanavalin A (Con A). Maximum capacity of transport (Vmax) at 37 degrees C and 136.9 mM Na+ was 30 pmol/10(6) cells/30 seconds, while the apparent Km was 142 microM. In cells exposed to 10 mM histidine, asparagine, serine, or leucine transport of glutamine declined to 28%, 15%, 17%, and 21%, respectively, of the rates in controls. Inhibition by histidine (Ki = 0.58 mM) and serine (Ki = 0.25 mM) was competitive, by leucine was non-competitive (Ki = 0.64), while alpha-methylamino-isobutyric acid and 2-amino carboxy-bicyclo (2.2.1)-heptane had no effect. In cells cultured for 24 hours with or without 10 micrograms/ml Con A, the apparent Km was 70 microM vs. 89 microM and Vmax 73 vs. 26 pmol/10(6) cells/30 seconds. Sodium depletion (9.0 mM NaCl) greatly diminished glutamine transport in resting and stimulated cells. Inhibition of glutamine transport by serine was sodium sensitive, while inhibition by histidine and asparagine was not. Serine had no competitive effect in sodium-depleted media. The data demonstrate what appear to be two carrier systems for glutamine, sodium sensitive and sodium insensitive. It is suggested that glutamine transport into lymphocytes occurs via processes similar to System N and System ASC described in other cells, with System ASC as the sodium-sensitive component. Con A augments the capacity rather than the affinity of glutamine transporting systems.     

Early changes in concanavalin A-stimulated lymphocytes detected by the fluorescent probe N-phenyl-1-naphthylamine

Summary The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 g/ml concanavalin A (Con A) for 30 min at 37° C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by ³H-thymidine assay.Increased fluorescence was not observed in the presence of 10 mM -methyl mannoside, 5mM sodium azide, 10^–5 M cytochalasin B, or Ca²⁺-free culture medium.However, incubation with 10^–5 M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response.Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweed mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.Supported by a grant from the Anti-Cancer Council of VictoriaWe are grateful to Miss R. Jenkins and Mr. R. McGready for preparations of succinyl-Con A, to Dr. H.A. Ward for helpful discussion, and to Dr. M. Hohnes of the Walter and Eliza Hall Institute for providing BALB/c.nu mice     

Developmental changes in humoral suppressor activity released from concanavalin A-stimulated human lymphocytes on B cell differentiation

Cell-free culture supernatants (Con A-activated supernatants) were obtained by incubating peripheral blood lymphocytes (PBL) from cord blood, healthy children of various ages, and healthy adults with mitogenic doses of concanavalin A (Con A) for 48 hr. It is well known that human T lymphocytes are activated by Con A to manifest suppressor function in vitro. One mechanism whereby these suppressor cells act has been shown to be by the secretion of a soluble suppressor factor. The present study has investigated the Con A-inducible suppressor cell function in cord blood, children of various ages, and adults by comparing the ability of each Con A-activated supernatant to inhibit the generation of immunoglobulin-producing cells (Ig-PC) in pokeweed mitogen- (PWM) stimulated cultures of adult PBL. Con A-activated supernatants from adults could markedly suppress the generation of Ig-PC by allogeneic as well as autologous PBL in response to PWM. Such suppression appeared to be equally effective on the generation of IG-PC of 3 major classes, IgG, IgM, and IgA. On the contrary, Con A-activated supernatants from cord blood and newborn infants showed only a negligible suppression on PWM-induced adult B cell differentiation. But the suppressor activity found in Con A-activated supernatants gradually increased with advancing age, and reached approximately to the adult level at 4 yr of age or later. The results suggest that human T lymphocytes may be relatively deficient in their Con A-induced suppressor cell function in the early period of life.     

Enhanced turnover of arachidonic acid-containing species of phosphatidylinositol and phosphatidic acid of concanavalin A-stimulated lymphocytes

The incorporation of [32P]orthophosphate into phosphatidylinositol (PI) of pig lymphocytes was markedly increased by stimulation with concanavalin A. The labeling of PI with [3H]glycerol was also enhanced significantly, indicating that both de novo synthesis and recircular system (PI response) of PI were accelerated. This rapid labeling of PI might be related to the rapid breakdown of phosphatidylinositol 4,5-bisphosphate which was observed in various stimulated tissues. Concanavalin A also accelerated the labeling of phosphatidic acid with 32P and [3H]glycerol. To determine the dependence of this phenomenon on the fatty acid composition of both phospholipids, we separated PI and phosphatidic acid into individual molecular species. The predominant molecular species in PI was tetraene (81.6%) and those in phosphatidic acid were monoene (53.0%), diene (15.8%) and tetraene (19.2%), respectively. Interestingly, the incorporation of 32P into arachidonic acid-containing species (tetraene) was most rapidly elevated. On the other hand, the increment of 32P into saturated + monoene, diene and triene was relatively smaller and resembled that of [3H]glycerol. Similarly, the incorporation of 32P into tetraene of phosphatidic acid was preferentially accelerated. This is the first report concerning the metabolism of molecular species of phosphatidic acid in stimulated cells. These results indicate that the PI recirculating system is virtually dependent on tetraenoic species and that the participation of other molecular species is small. The increased de novo synthesis mainly depends upon molecular species other than tetraene. Arachidonic acid-containing species which turn over rapidly via the PI cycle may have an important role in the mitogenic triggering.     

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate on concanavalin A-stimulated sterol and fatty acid synthesis in mouse spleen lymphocytes.

Substantial increases in both 3beta-OH sterol and fatty acid synthesis were observed after concanavalin A addition to mouse spleen lymphocytes cultured in serum-free media. The rate of sterol synthesis increased linearly up to 60 h. The rate of fatty acid synthesis increased up to 20 h, reaching a plateau in synthetic activity which was the maintained. CO2 production from acetate was slightly stimulated by concanavalin A. In contrast to sterol and fatty acid synthesis, the rate of CO2 production in both mitogen-stimulated and resting cultures declined with time. Dibutyryl cyclic AMP had a strong inhibitory effect on concanavalin A-stimulated sterol and fatty acid synthesis from acetate, but only a slight effect on CO2 production. Delayed addition of dibutyryl cyclic AMP resulted in reduced inhibition. The data suggest a sequence of initiation for fatty acid and sterol synthesis prior to DNA synthesis and a possible regulatory role of cyclic AMP in this initiation. The results support the hypothesis that lymphocyte activation is sequential within the spleen cell population and is accompanied by fatty acid and sterol synthesis.