The suppressive effect of gangliosides upon IL 2-dependent proliferation as a function of inhibition of IL 2-receptor association

Gangliosides inhibited the proliferation of mitogen-activated human peripheral blood lymphocytes and the IL 2-dependent growth of murine T cell lines and 5-day-old human PHA lymphoblasts. In the case of the murine cell lines and PHA lymphoblasts, most of the effect of gangliosides could be reversed by the addition of high levels of IL 2. In the case of freshly-stimulated mitogen blasts, however, the ganglioside-induced inhibition could not be reversed by increasing exogenous IL 2 levels. These results indicate that inhibition of proliferation by gangliosides can be divided into IL 2-reversible and IL 2-irreversible mechanisms, the latter of which were predominant during the initial stage of cellular activation. Inclusion of gangliosides in receptor binding assays for radiolabeled IL 2 indicated that the IL 2-reversible mechanism likely involved competition between gangliosides and the cellular receptor for the binding of IL 2. Gangliosides blocked binding of radiolabeled IL 2 to both the high and low affinity forms of the IL 2 receptor, and this effect was most noticeable when the gangliosides and IL 2 were preincubated before addition of the target cells. In contrast, treatment of cells with gangliosides had no effect on the affinity of the cellular IL 2 receptor if the free gangliosides were removed immediately before the binding assay. Gangliosides also blocked the binding of radiolabeled IL 2 to anti-IL 2 antibodies, supporting the notion that their inhibitory effect is mediated via a direct interaction with IL 2. Thus, one major mechanism by which gangliosides block the IL 2-dependent proliferation of activated cells is by the sequestering or inactivation of the IL 2 molecule. This effect is reversible with the addition of excess IL 2, which distinguishes it from other mechanisms of ganglioside-dependent inhibition operating during the cellular activation process.     

Studies of the mechanism by which gangliosides inhibit the proliferative response of murine splenocytes to concanavalin A

Gangliosides are known to inhibit the proliferative response of murine and human lymphocytes to antigens and mitogens in vitro. In this study the response of murine spleen cells to concanavalin A (Con A) was used as a model system. Analysis of the cellular events by flow cytometry revealed that during the first 24 hr of culture the effect of gangliosides on Con A-treated cells was minimal. At 48 hr, however, more of the ganglioside-treated cells were in G0/G1, the cells contained more RNA, and fewer cells were in S phase. These data indicate that gangliosides inhibit the transition of the cells from G0/G1 into the S phase of the cell cycle. Expression of the interleukin 2 (IL-2) receptor, as measured by the binding of a monoclonal antibody to the receptor, was not inhibited by the gangliosides. Binding of 125I-labeled recombinant IL-2 to cells cultured for 48 hr with Con A was inhibited by ganglioside GD1a but not by asialo GM1. Inhibition was much more effective if the gangliosides were preincubated with IL-2 before addition of cells, but no inhibition was observed if the cells were preincubated with gangliosides and the unbound gangliosides were washed out prior to addition of the IL-2. These data suggest that interference with the binding of IL-2 to the high-affinity IL-2 receptor of activated T lymphocytes plays an important role in the inhibition of Con A-induced proliferation.     

VLDL modulates the cytokine secretion profile to a proinflammatory pattern.

Human very-low-density lipoprotein (VLDL) inhibits DNA synthesis in lymphocytes activated by the nonspecific mitogen concanavalin A (Con A). We studied the effects of VLDL on lymphocyte activation (IL-2 receptor expression), cell cycle progression, and production of IL-2 and of IL-4 (a proinflammatory and an anti-inflammatory interleukin, respectively) to understand why an atherogenic lipoprotein inhibits cell proliferation. After 48 h of stimulation with the mitogen, VLDL decreased the population of cells bearing IL-2 receptor and the population of T-cells that progress through the cell cycle, increasing the population of T-cells in G(0)/G(1). Cells cultured in the presence of Con A and VLDL produced higher levels of IL-2 and lower levels of IL-4 than cells cultured without VLDL. These results suggest that VLDL inhibits lymphocyte proliferation by reducing IL-2 receptor and enhancing the levels of IL-2. Probably, one atherogenic effect of VLDL is to modulate the cytokine secretion profile of lymphocytes to a predominantly proinflammatory response.     

Glycophorin A interacts with interleukin-2 and inhibits interleukin-2-dependent T-lymphocyte proliferation.

Sialoglycolipids shed by tumor cells have been implicated in tumor-induced inhibition of T-lymphocyte responses to interleukin-2 (IL-2). In the present study, we have used glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, to investigate whether shedding of glycoproteins might also contribute to immunosuppression. Glycophorin A inhibited IL-2-stimulated proliferation of the IL-2-dependent cell lines HT-2 and CTLL-2 in a dose-dependent manner. Time course studies on synchronized cell populations indicated that the glycoprotein acted early in the activation process. On the other hand, glycophorin A had essentially no effect on IL-1-mediated stimulation of the IL-1-sensitive thymocyte cell line EL-4 NOB-1. Gel filtration FPLC demonstrated that IL-2 was able to bind to glycophorin aggregates under physiological conditions. Reconstituted vesicles containing glycophorin were also shown to bind IL-2. In addition, both soluble glycophorin aggregates and lipid vesicles containing glycophorin blocked binding of IL-2 to high-affinity cellular IL-2 receptors. Taken together, these results suggest that shedding of tumor sialoglycoproteins with oligosaccharide chains similar to glycophorin A might contribute to negative modulation of IL-2-mediated immune responses.     

Gangliosides suppress the proliferation of autoreactive cells in experimental allergic encephalomyelitis: ganglioside effects on IL-2 activity

Gangliosides have been shown to suppress human and murine lymphocyte proliferative responses in vitro. We tested the suppressive effects of gangliosides on the proliferation of autoreactive lymphoid cells obtained from Lewis rats with experimental allergic encephalomyelitis (EAE). Exogenous rat brain gangliosides inhibited both antigen- and mitogen-induced proliferation by as much as 79 and 93%, respectively. Gangliosides similarly inhibited the antigen-induced proliferation of a myelin basic protein (MBP)-reactive T-cell line which is able to passively induce EAE. Suppression was greatest when gangliosides were added at the initiation of culture, and was not abrogated by supraoptimal antigen concentration. Interleukin 2 (IL-2) activity in culture supernatants was not diminished by the addition of gangliosides. Gangliosides did not inhibit the IL-2-induced proliferation of a murine IL-2-dependent cell line, CTLL-20, unless the IL-2 was first preincubated with gangliosides before the addition of CTLL-20. Preincubation of CTLL-20 with gangliosides resulted in no inhibition of the subsequent responses to IL-2. Exogenous gangliosides did not decrease the binding of a monoclonal antibody directed against the rat cell surface IL-2 receptor. Addition of exogenous IL-2 to ganglioside-suppressed cultures had no effect or only partially restored the proliferative responses. Therefore, gangliosides were shown to inhibit the proliferation of autoreactive lymphoid cells without affecting IL-2 production or IL-2 receptor expression.     

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

Membrane gangliosides modulate interleukin-2-stimulated T-lymphocyte proliferation.

Membrane gangliosides appear to modulate signal transduction by several growth factor receptors. We have investigated the possible regulation of IL-2-induced proliferation signals by gangliosides. Low concentrations of cholera toxin B subunit (CT-B), which binds specifically to GM1 ganglioside, greatly inhibited IL-2-stimulated DNA synthesis in the IL-2-dependent cell line CTLL-2, but had no effect on proliferation of HT-2. GM1 levels proved to be very low in HT-2 compared to CTLL-2. Large increases in membrane-associated GM1 could be achieved in both cell lines by incubation with exogenous GM1, resulting in a high degree of inhibition of proliferation by CT-B for both CTLL-2 and HT-2. Inhibition was blocked by large unilamellar vesicles containing GM1, but not by vesicles of lipid alone. The time course of CT-B inhibition for CTLL-2 synchronized in G0-G1, indicated that the negative growth signal acts relatively early in the IL-2 activation pathway. CT-B did not affect binding of IL-2 to high-affinity IL-2r. The inhibitory effects of CT-B could not be reversed by pertussis toxin, suggesting that a G protein is probably not involved. These results show that CT-B binding to either endogenous or inserted GM1 can modulate IL-2-induced lymphocyte proliferation.     

Residual activation events functional after irradiation of mouse splenic lymphocytes

The radioresistance of lymphocytes increases after mitogenic stimulation, suggesting that a radiosensitive activation event contributes to the overall radiosensitivity of lymphocytes. We have sought to identify this activation event by determining the extent of activation of mitogen-stimulated lymphocytes previously exposed to growth-inhibiting doses of radiation. Mouse splenic lymphocytes were exposed to 0-15 Gy 137Cs radiation, and structural and functional damage were assayed. Although damage to cellular thiols and nonprotein thiols was modest, there was a significant loss of viability by 6 h as determined by uptake of propidium iodide (PI). Since cells did not die immediately after irradiation, the activation events which remained were evaluated. Growth-inhibiting doses of radiation left cells partially responsive to mitogen, in that cells were able to exit G0 phase, but they could progress no further into the cell cycle than G1a phase. It is important to note that assessment of viability by uptake of PI indicated substantial cell death after 15 Gy (45%, 6 h; 90%, 24 h); however, cell cycle analysis at 24 h indicated no significant decrease in progression from G0 to G1a phase. The LPS-stimulated response of B cells was more radiosensitive than the Con A-stimulated response of T cells. Further analysis of the Con A response indicated that production of interleukin-2 (IL-2) was unaffected, but expression of the IL-2 receptor was inhibited. Inhibition of poly-ADP-ribosylation and damage to lipids did not prevent the lack of mitogen responsiveness, since neither the ADP-ribose transferase inhibitor 3-aminobenzamide nor lipid radical scavengers had restorative effects on the mitogenic response. Nor was Con A-stimulated incorporation of [3H]thymidine restored with inhibitors of prostaglandin or leukotriene synthesis, suggesting that inhibition was due to direct effects on the Con A responders, and not indirect effects mediated by arachidonate metabolites. These results indicate that growth-inhibiting doses of radiation trigger the process in lymphocytes that culminates in apoptosis, yet leave the cells partially responsive to mitogenic stimuli.     

Interleukin-13 secretion by normal and posttransplant T lymphocytes; in vitro studies of cellular immune responses in the presence of acute leukaemia blast cells

T lymphocyte secretion of interleukin-13 (IL-13) in response to different activation signals was characterized in vitro. IL-13 release was investigated when virus transformed B lymphocytes or acute myelogenous leukaemia (AML) blasts were used as accessory cells during T cell activation. First, a majority of both CD4⁺ and CD8⁺ TCRαβ⁺ T lymphocyte clones, derived from normal individuals and bone marrow transplant recipients, secreted IL-13 in response to a standardized mitogenic activation signal (phytohaemagglutinin+IL-2+ B lymphocyte accessory cells). The CD4⁺ cells showed significantly higher IL-13 levels than the CD8⁺ subsets. Second, when leukaemic accessory cells (more than 95% AML blasts) were used during T cell activation, IL-13 was released both during alloactivation of normal T lymphocytes and during mitogen activation of posttransplant T cells. Third, when normal T lymphocytes were stimulated with allogeneic AML blasts, addition of IL-13-neutralizing monoclonal antibodies decreased interferon γ levels. Although addition of IL-13-neutralizing antibodies did not alter granulocyte-colony-stimulating factor secretion by allostimulating AML blasts, altered blast proliferation was detected for certain patients. Thus, most T cell clones can release IL-13, and IL-13 can modulate cytokine responses during T cell recognition of allogeneic AML cells. Received: 24 April 1997 / Accepted: 24 July 1997     

Human peripheral lymphocyte growth regulation and response to phorbol esters is linked to synthesis and phosphorylation of the cytosolic protein, prosolin

Prosolin is a major cytosolic protein (Mr 18400, isoelectric point 5.9) first reported in HL-60 promyelocytic leukemia cells. It is rapidly phosphorylated (15 to 30 min) in response to TPA treatment as an early event in a sequence that leads to cessation of cell proliferation and to differentiation of promyelocytes into monocytes. In our study we examined the expression of prosolin in human peripheral lymphocytes and investigated the effects of TPA treatment on prosolin phosphorylation and on lymphocyte proliferation. Prosolin was not expressed in resting PBL but was induced after 24 to 36 h of PHA stimulation, simultaneously with induction of DNA synthesis. In rapidly proliferating (IL-2 dependent) PBL prosolin was a major cytosolic component, comprising 0.5% of total cytosolic protein, of which approximately 28% was phosphorylated. Expression of prosolin decreased again when either mitogen-induced or IL-2-dependent proliferation diminished during extended periods in culture. Thus, expression of prosolin is correlated with periods when PBL are cycling through S-phase. TPA treatment of IL-2-dependent PBL at the peak of their growth caused phosphorylation of about two-thirds of preexisting unphosphorylated prosolin within 1 h. This was accompanied by cessation of cell proliferation, as indicated by measurements of TdR incorporation. Although TPA has well known mitogenic effects in lymphocytes during initial activation, this result shows that it exerts an antiproliferative effect in rapidly dividing PBL. It is suggested that increased phosphorylation of prosolin may be an initiating event in the antiproliferative response to TPA, which would occur only in proliferating lymphocytes expressing prosolin.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

Characterization of a low molecular weight suppressor of lymphocyte proliferation from guinea pig L2C leukemia cells

Conditioned medium (CM) from 24-hr culture of guinea pig L2C B lymphoblastic leukemia cells contained an inhibitor(s) of mitogen- and antigen-stimulated proliferation of syngeneic (strain 2 guinea pigs), allogeneic (Hartley guinea pigs), and xenogeneic (Balb/c mouse, NZW rabbit) lymphocytes. The proliferation of several lymphoid and nonlymphoid cell lines also was inhibited in the presence of CM. The inhibitor(s) in CM was not toxic to any of the cultures studied. CM inhibited the mitogen-stimulated proliferation of lymphocytes when added to cultures up to 52 hr after addition of mitogen. Normal responsiveness to mitogens could be restored by washing the CM-treated lymphocytes with medium during the first 6 hr of culture. The addition of exogenous IL-2 to lymphocyte cultures did not overcome the CM-mediated suppression of mitogen- or antigen-stimulated proliferation. CM also inhibited the IL-2-dependent proliferation of murine CTLL-2 cells. Preincubation of guinea pig lymphocytes in CM did not inhibit the capacity of these cells to release IL-2 after exposure to mitogen. The antiproliferative activity of CM was stable to heating at low pH (100 degrees C, 10 min, pH 4.0), was resistant to treatment with papain, pronase, DNase, and RNase and did not bind to Con A-Sepharose. Incubation of the L2C cells in indomethacin did not inhibit the release of the inhibitor(s). The inhibitor(s) in CM had an apparent molecular weight of 500-3500 Da as determined by dialysis and ultrafiltration analysis. The inhibitory activity was recovered in the organic phase after extraction with chloroform:methanol and eluted distinct from the thymidine standard after gel filtration on Sephadex-G 25. These data suggest that the inhibitor(s) in CM is a nonspecific, low molecular weight, lipid-like component (not prostaglandin) that exerts its antiproliferative effects subsequent to cell activation. The inhibitor(s) did not appear to suppress other biologic functions associated with activation, such as IL-2 secretion. The inhibitor in CM may be important in promoting tumor survival in vivo by suppressing potential anti-tumor cellular immune responsiveness.     

Expression of interleukin 2 and the interleukin 2 receptor in aging rats

Lymphocytes of aged animals exhibit a marked decrease in proliferative capacity in response to mitogen stimulation when compared to those of younger animals. In humans and mice the decreased proliferation is due at least in part (i) to the inability of lymphocytes to synthesize sufficient interleukin 2 (IL-2) and (ii) to decreased expression of IL-2 receptors (IL-2R) on the surface of aged lymphocytes. We compared proliferative abilities, IL-2 production, and IL-2R expression in splenocyte cultures of 4- to 5- and 22- to 24-month-old Fischer 344 rats stimulated with either concanavalin A (Con A) or A23187 and phorbol myristate acetate (PMA). Proliferation was significantly decreased in aged lymphocytes (30-50%) with both treatment protocols. However, unlike mice and humans we observed no difference in IL-2 activity, IL-2 mRNA levels, or IL-2R cell surface expression of lymphocytes from young and aged rats stimulated with either Con A or A23187 and PMA. These results indicate that factors other than decreased expression of IL-2 and IL-2R are responsible for the diminished proliferative capacity of aged rat lymphocytes following mitogen stimulation.     

Agents that mimic antigen receptor signaling inhibit proliferation of cloned murine T lymphocytes induced by IL-2

We have shown previously that stimulation of cloned murine T lymphocytes via the TCR inhibits their responsiveness to rIL-2. Signaling via the TCR is believed to result in a variety of biochemical events that include a rise in intracellular free calcium and activation (translocation) of protein kinase C. These two signals also can be generated by calcium ionophores, such as ionomycin, and by activators of protein kinase C, such as PMA. We report here that treatment of cloned murine T lymphocytes with PMA, ionomycin, or the combination led to a dose-dependent inhibition of IL-2-dependent proliferation but did not inhibit lymphokine secretion. Concentrations of PMA and ionomycin that maximally inhibited proliferation stimulated maximal lymphokine secretion and increased mitochondrial activity as assessed by measurement of cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide. Furthermore, PMA, ionomycin, the combination, or immobilized anti-CD3 mAb added after 12 to 16 h of culture with IL-2 could inhibit proliferation. These results demonstrate that PMA and ionomycin mimic stimulation of the TCR by high concentrations of immobilized anti-TCR mAb in that proliferation is inhibited and lymphokine secretion is induced. In addition, PMA or ionomycin could independently inhibit proliferation of some cells. These findings suggest that alternative mechanisms exist to regulate proliferation. Either increased levels of intracellular calcium or the physiologic events corresponding to those induced by PMA can inhibit IL-2-dependent replication of T lymphocytes.     

Pre-exposure of human B cells to recombinant IL-1 enhances subsequent proliferation

The reported effects of the monocyte-derived cytokine IL-1 on human B lymphocytes are both varied and controversial. IL-1 has been reported to augment both proliferation and Ig secretion of previously activated human B cells. In the present study highly purified splenic B cells were cultured with rIL-1 before, simultaneously with, and after the addition of the polyclonal B cell mitogen, anti-Ig. rIL-1 had no significant effect on B cell proliferation when added simultaneously with or after B cell activation with anti-Ig. However, incubation of splenic B cells with rIL-1 for 24 h before stimulation with anti-Ig appeared to enhance mitogenesis. With the observation that rIL-1 exerted effects on resting B cells, the effect of rIL-1 on several events which accompany B cell activation was examined. rIL-1 failed to stimulate RNA synthesis, effect increases in cell size or intracellular Ca2+ levels, or lead to the hyperexpression of MHC class II or B cell activation Ag. These studies suggest that rIL-1 does not activate B cells but primes them to respond to subsequent activation.     

Modulation of IL-2- and IL-4-dependent human B cell proliferation by cyclic AMP.

The second messenger cAMP is a modulator of cellular growth possessing both inhibitory and stimulatory properties. In this report, we show that IL-2- and IL-4-dependent DNA synthesis of anti-mu-activated human B cells is modulated in opposite ways by agents increasing intracellular levels of cAMP. Forskolin and 2'-O-dibutyriladenosine-3',5'-cyclic monophosphate had no proliferative effect by themselves. Nevertheless they decreased IL-2-driven proliferation and increased IL-4-mediated DNA synthesis. IL-4 and cAMP each inhibited the IL-2-dependent proliferation with similar patterns of reactivity. Both IL-4 and forskolin needed to be present during the first 48 h of culture to display inhibitory activity, and preactivation of B cells for 16 h with forskolin and IL-4 did not prevent further B cell response to IL-2. This suggests that cAMP and IL-4 directly interact with IL-2 signaling. In addition, we show that the cAMP-dependent protein kinase inhibitor N-(2-methylamino-ethyl)-5-iso-quinoline-sulfamide reversed the IL-4-inhibitory effect on IL-2-driven proliferation. Our data suggest that the IL-4-inhibitory signal to IL-2-driven human B cell proliferation involves cAMP-dependent protein kinase activation.     

Transforming growth factor-beta s are equipotent growth inhibitors of interleukin-1-induced thymocyte proliferation

The effects of two forms of transforming growth factor-beta, TGF-beta 1 and TGF-beta 2, upon the proliferative response of murine thymocytes were investigated in this study. TGF-beta 1 and TGF-beta 2 were found to be equipotent growth inhibitors of interleukin-1 (IL-1)- and phytohemagglutinin (PHA)-stimulated thymocytes when added at the initiation of the cultures. These factors suppressed the proliferative response in a dose-dependent fashion between 0.4 and 100 pM. The proliferative response was maximally inhibited (90% inhibition) at 100 pM. The half-maximal inhibitory dose (ID50) was 6 and 4 pM for TGF-beta 1 and TGF-beta 2, respectively. These factors were less effective or ineffective at suppressing the proliferation of thymocytes which had been prestimulated for 24 to 48 hr by IL-1 and PHA. Neither factor inhibited interleukin-2 (IL-2)-dependent thymocyte proliferation or the proliferation of an IL-2-dependent cytotoxic T cell line (CTL-L), suggesting that the anti-proliferative actions of these factors was by inhibition of cellular events triggered by IL-1. Furthermore, anti-TGF-beta 1 antibodies did neutralize the biological actions of TGF-beta 1 and these antibodies did block the binding of 125I-labeled TGF-beta 1 to cell surface receptors showing that the inhibitory action is mediated through specific receptors for TGF-beta 1 on thymocytes. These antibodies, however, did not neutralize the anti-proliferative action of TGF-beta 2. Although TGF-beta 1 and TGF-beta 2 exhibit very similar biological activities, these molecules are antigenically different and, therefore, have different tertiary structures.