Role of lipomodulin, a phospholipase inhibitory protein, in immunoregulation by thymocytes

The treatment of murine thymocytes with anti-lipomodulin antibody during Con A stimulation causes selective loss of suppressor activity, but not of helper activity on PFC assay, when co-cultured with T cell-depleted spleen cells. Interaction of the antibody with responder cells in thymocyte culture were necessary in the early stage rather than in the later stage of lymphocyte activation by Con A, which suggests that anti-lipomodulin antibody acts in the stage of suppressor T cells generation. When thymocytes were cultured with purified lipomodulin for 48 hr, suppressor activity was induced. Lipomodulin as detected by radioimmunoassay was found to be released from T cells with the phenotype of I-J+, Lyt-1-, Lyt-2+. The immunoprecipitates from the media of Con A-stimulated thymocyte with anti-I-Kk antibody and anti-lipomodulin antibody were analyzed on SDS-gel electrophoresis. I-J products had m.w. 36,000 and 24,000, whereas lipomodulin had m.w. 36,000, 24,000, and 15,000. Because anti-I-Jk antibody could precipitate 125I-labeled lipomodulin purified from rabbit neutrophils, these results suggest that lipomodulin is a product of I-J genes that induces suppressor T cells.     

Modulation of lymphocyte proliferation by macrophages and macrophages loaded with arachidonic acid

Arachidonic acid (AA) is incorporated and exported by macrophages. This fatty acid is also transferred from macrophages (Mphi) to lymphocytes (LY) in co-culture. This observation led us to investigate the effect of macrophages pre-loaded with AA on concanavalin A (Con A)-stimulated lymphocyte proliferation. The experiments were performed in co-culture. This condition reproduces the in vivo microenvironment in which the modulation of lymphocyte proliferation is dependent on the interaction with macrophages. Lymphocytes obtained from untreated rats or from intraperitoneally thioglycolate-injected rats (THIO-treated) were co-cultured with macrophages from the same rats. Firstly, macrophages were co-cultured for 48 h with Con A-stimulated lymphocytes in different proportions: 0.5, 1, 2.5, 5, 10, 20 and 30% of 5 x 10(5) lymphocytes per well. At 1% proportion, macrophages caused maximum stimulation of lymphocyte proliferation; a four- to five-fold increase, for cells from both thioglycolate-treated and untreated rats, respectively, whereas at 20% it caused maximum inhibition. In addition, 1 or 20% macrophages were pre-loaded with several AA concentrations during a period of 6 h and co-cultured with lymphocytes. At 180 microM AA and 1% macrophages, lymphocyte proliferation was inhibited (by 25%), whereas at 20% macrophages, proliferation was increased, by 25- and three-fold, respectively, for cells from untreated and THIO-treated rats. AA added directly to the medium reduced lymphocyte proliferation, also being toxic to these cells at 100 microM. No toxic effects of AA were observed on macrophages. Additional evidence suggests that nitric oxide production is involved in the modulation of lymphocyte proliferation by AA-pre-loaded macrophages. These findings support the proposition that AA can directly modulate lymphocyte proliferation and the interaction between macrophages and lymphocytes.     

The effect of beta-estradiol, progesterone and testosterone on the production of human leukocyte derived interferons

Sex hormones including estrogens, progesterone and testosterones are known to have adverse effects on the immune system and particularly on the proliferative response. Since cytokine production is known to be dissociable from the proliferation of lymphocytes and since other steroid hormones profoundly affect cytokine production, we felt it would be important to know the effect of sex steroids on the production of interferons (IFN), particularly since the latter are known to be key substances in the immune response. We have shown estradiol can slightly reduce gamma IFN yields with certain inducers (Con A, SEA) but only in pharmacologic concentrations. Similarly, progesterone had a modest effect in the same concentrations but only when Con A was the inducer. Testosterone did not effect IFN titers at any concentration. None of the sex steroids affected alpha IFN production and none of them influenced the bioactivity of either IFN species. In all cases these hormones diminished proliferative responses as has been previously noted.     

The alveolar macrophage: Its capacity to act as an accessory cell in mitogen-stimulated proliferation of guinea pig lymphocytes

The capacity of the alveolar macrophage to act as an accessory cell in PHA-induced lymphocyte proliferation was investigated and compared with that of the peritoneal and peritoneal exudate macrophages in guinea pigs. When lymph node cells were co-cultured with autologous lung cells recovered by airway lavage, the proliferative response to PHA was greatly enhanced over that of lymph node cells alone. In the presence of peritoneal cells or peritoneal exudate (glycogen-induced) cells, the PHA response was intermediate between that of lymph node cells alone and lymph node cells cultured with lung cells. Experiments using purified macrophages (≥98%) as accessory cells demonstrated that the difference observed between lung and peritoneal accessory cells was due to differences in macrophage function. Furthermore, when lymph node cells were cultured in the upper chamber of a double-chambered Marbrook apparatus, PHA-induced proliferation was enhanced only when lung and not peritoneal macrophages were present in the lower chamber. Additional experiments showed that this difference (1) was not an artifact of the thymidine incorporation assay to measure proliferation; (2) was not affected by changing the macrophage-lymphocyte ratio; and (3) was not simply a trephocytic or growth promoting effect of macrophages which could be replaced by 2-mercaptoethanol.These findings show that macrophages from different sources differ in their abilities to act as accessory cells in PHA-induced lymphocyte proliferation. Alveolar macrophages appear to have an enhanced capacity compared to unstimulated and stimulated peritoneal macrophages in this function. At least part of this difference may be due to a difference in the elaboration of soluble factor (s) by macrophages.     

Regulation of T cell proliferation by cloned interferon-alpha mediated by Leu-11b-positive cells

The autologous T lymphocyte proliferative response (AMLR) induced by a B lymphocyte-enriched non-T, nonadherent cell population (NT, NAC) and by a macrophage-enriched population were both suppressed by the addition of a cloned interferon-alpha (IFN-alpha Con1) directly to the cultures. Preincubation of the stimulating NT, NAC with IFN-alpha Con1 resulted in comparable suppression. In contrast, preincubation of the macrophages with IFN-alpha Con1 resulted in significant augmentation of T cell proliferation. Depletion of Leu-11b-positive cells from the NT, NAC exposed to IFN-alpha Con1 restored the autologous T cell response. Addition of IFN-alpha Con1 activated Leu-11b-positive cells, isolated from the NT, NAC population, was suppressive of the AMLR. Although NK cytotoxicity was irradiation sensitive, suppression of the AMLR by IFN-alpha Con1-activated NT, NAC was resistant, suggesting that different subsets of cells or mechanisms by the same cells may have been responsible. These observations may offer insights into the potential role of cells with the NK phenotype, Leu-11b, and IFN in contributing to immuno-regulatory changes observed in clinical states associated with elevated concentrations of IFN.     

Humoral factors and nonspecific immune suppression in Syrian hamsters infected with Leishmania donovani

Leishmania donovani produces progressive wasting and ultimately fatal visceral leishmaniasis in Syrian hamsters and provides an excellent model of progressive disease in humans. Experimentally infected hamsters were used to investigate the development of nonspecific immune suppression during visceral leishmaniasis and its association with humoral factors and wasting. At 2 wk all infected hamsters had developed antibody against a 59-kDa parasite antigen not recognized by sera of control hamsters. By 4 wk, strong antibody responses were noted against antigens of 26, 35, 46, 69, 107, and 120 kDa. No additional antigen was recognized at 6 or 8 wk or in hamsters treated with high doses of a pentavalent antimonial (stibogluconate sodium, 100 mg/kg/day for 5 days). Weight loss was first noted in infected hamsters at 8 wk. No difference in splenic lymphocyte proliferation in response to concanavalin A (Con A) was noted at 2 wk, but by 6 wk infected animals had only 20% of the Con A response of controls, and by 8 wk only 13%. Furthermore, incubation of splenic lymphocytes from uninfected control animals with 5% fetal calf serum and 5% serum from infected hamsters obtained at 4, 6, or 8 wk suppressed Con A responses by 50%, 99%, and 100%, respectively. Spleen cells from drug-treated animals exhibited no suppression of Con A responses when incubated with 5% autologous serum, but there was profound suppression when they were incubated with 5% autologous serum obtained during the acute phase of infection. Humoral factors, but not wasting, contributed to the suppression of lymphocyte responses.     

Accessory cell function of cells of the mononuclear phagocyte system isolated from mycobacterial granulomas

Epithelioid cells from BCG-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas were examined for their ability to act as accessory cells for T-cell proliferation to mitogen (Con A) and antigen (PPD). The granuloma cells were separated on a FACS using monoclonal antibody specific to guinea pig macrophages. Epithelioid cells (which are Ia negative) were able to support proliferation to Con A but not to antigen. Cultures containing Ia positive granuloma macrophages from M. leprae sensitized animals did not show responsiveness to Con A or to PPD. Oil-induced peritoneal exudate macrophages from BCG or M. leprae immunized animals were able to act as accessory cells for both mitogen and antigen proliferation. The nonresponsiveness of cultures containing epithelioid cells stimulated with PPD or M. leprae granuloma macrophages stimulated with Con A was not due to suboptimal or supraoptimal accessory cell:lymphocyte ratios.     

In vitro production of immune interferon (IFN gamma) by murine spleen cells when different sensitizing antigens are used in vivo and in vitro

OK-432, a killed preparation of Streptococcus pyogenes, as well as bacillus Calmette-Guérin (BCG) and Corynebacterium parvum are all known to induce immune interferon (IFN gamma) in mice. To examine the mechanisms of IFN gamma induction by OK-432, DDI mice were sensitized with various doses of OK-432, either by a single injection of a 1-mg dose or repeated injections of 0.1-mg doses given intraperitoneally. Spleen cells removed from the mice 7-9 days after the last injection produced high-titered IFN gamma (600-800 IU/ml) in vitro in the presence of 5 micrograms/ml of OK-432. In the absence of OK-432, however, in vitro IFN gamma production of sensitized spleen cells was quite limited. Moreover, when inducers of different antigenic entities such as serologically unrelated Streptococcus faecalis, Listeria monocytogenes, or Con A were added in vitro, instead of OK-432, to the OK-432-sensitized spleen cells, high-titered IFN gamma production also occurred. This may indicate that the signal required by T cells to produce IFN gamma in vitro need not necessarily be the same as that required to sensitize mouse macrophage in vivo. Once sensitized with OK-432, mice spleen cells continued to produce high-titered IFN gamma for more than 3 but less than 5 weeks.     

Production of gamma interferon by human T and null cells and its regulation by macrophages

Human mononuclear subpopulations were tested for the capacity to produce interferon after mitogenic stimulation with protein A from Staphylococcus aureus. Mononuclear cells were separated into highly enriched macrophage, T-, B-, and null-cell subpopulations by Sephadex G-10 adherence, anti-human IgG F(ab′) two-column chromatography, and rosetting with sheep erythrocytes. Interferon (IFN) production was observed in both T- and null-cell preparations, but not in macrophage or B-cell preparations. Physicochemical and antigenic characterization of IFN from T- and null-cell preparations showed that both mononuclear subpopulations produced gamma IFN (IFNγ). Regulatory studies showed that IFNγ production was differentially regulated by macrophages. Macrophage addition to T lymphocytes augmented both cellular proliferation and IFNγ production, whereas macrophage addition to null cells suppressed IFNγ production and had no effect on the minimal proliferative response observed for these cells.     

Effect of wheat germ agglutinin on the interleukin pathway of human T lymphocyte activation

Wheat germ agglutinin (WGA) inhibits proliferation of human peripheral blood mononuclear cells (PBMC) induced by mitogens and antigens. We investigated the mechanism by which WGA inhibits PHA-induced human lymphocyte proliferation with regard to the interleukin pathway. Our data revealed that although PBMC-proliferation was markedly suppressed by WGA, levels of IL 2 activity in WGA-inhibited cultures were not reduced, but instead were increased, suggesting failure to utilize IL 2. Furthermore, the addition of exogenous IL 2 failed to overcome the suppression. Consistent with these observations, culturing PBMC with PHA plus WGA markedly decreased the number of high-affinity IL 2 receptor per cell, as determined by binding of purified [3H]IL 2, relative to cultures containing PHA alone. WGA immobilized on support beads bound detergent-solubilized IL 2 receptors from PHA-activated T cells, but did not bind human IL 2. However, WGA did not competitively block the binding of [3H]IL 2 to PHA-induced lymphoblasts. These results suggest that WGA inhibits lymphocyte proliferation by binding to and decreasing the number of high-affinity IL 2 receptors displayed on T cells, without impairing IL 2 production.     

Regulation of lymphocyte proliferation by uterine serpin: interleukin-2 mRNA production, CD25 expression and responsiveness to interleukin-2

During pregnancy, the endometrium of the ewe secretes large amounts of a progesterone-induced protein of the serpin superfamily of serine proteinase inhibitors called ovine uterine serpin (OvUS). This protein inhibits lymphocyte proliferation in response to concanavalin A (ConA), phytohemagglutinin (PHA), or mixed lymphocyte reaction. The purpose of these experiments was to characterize the mechanism by which OvUS inhibits lymphocyte proliferation. Ovine US caused dose-dependent inhibition of lymphocyte proliferation induced by phorbol myristol acetate (PMA), an activator of protein kinase C. The PHA-induced increase in CD25 expression was inhibited in peripheral blood mononuclear leukocytes (PBML) by OvUS. However, no effect of OvUS on Con A-induced expression of CD25 was observed. Further analysis using two-color flow cytometry revealed that OvUS inhibited ConA-induced expression of CD25 in gammadelta-TCR- cells but not gammadelta-TCR+ cells. Stimulation of PBML for 14 hr with ConA resulted in an increase in steady state amounts of interleukin-2 (IL-2) mRNA that was not inhibited by OvUS. Ovine US was also inhibitory to lymphocyte proliferation induced by human IL-2. Results suggest that OvUS acts to inhibit lymphocyte proliferation by blocking the upregulation of the IL-2 receptor and inhibiting IL-2-mediated events. Lack of an effect of OvUS on ConA-stimulated CD25 expression in gammadelta-TCR+ cells may reflect a different mechanism of activation of these cells or insensitivity to inhibition by OvUS.     

Generation of suppressor cells by concanavalin A: a new perspective

Significantly lower mitogenic responses of fresh cells co-cultured with Con A-stimulated cells were found when compared with the responses of fresh cells co-cultured with preincubated control cells. We do not agree with the interpretation that this effect represents the generation of suppressor cells by Con A, since the responses of fresh cells cultured alone were also significantly less than when co-cultured with control cells and the same as when co-cultured with the Con A-stimulated cells. Treatment with mitomycin C was sufficient to prevent the preincubated cells from contributing to the mitogenic response of the fresh cells. The increased responses of fresh cells when co-cultured with preincubated cells seems analagous to the increased mitogenic responses of cells aged in vitro by preincubation without mitogen. This effect seems to be transferable to fresh cells in the absence of cell division. Although preincubation in the presence of Con A abrogates this effect, we do not interpret this as the generation of suppressor cells.     

Altered proliferative kinetics in PHA-activated human T-lymphocytes treated with the anti-HLA class I monoclonal antibody 01.65

Abstract. Anti-HLA class I monoclonal antibody (mAb) 01.65 inhibited phyto-haemagglutinin (PHA)-induced human lymphocyte proliferation. The inhibitory effect was inversely correlated to the strength of the proliferative response. It was increased when lymphocytes were stimulated with suboptimal doses of PHA but it disappeared with supraoptimal doses. Proliferation inhibition was achieved by prolonging the cell cycle time and by slowing down its recruitment rate. The former effect was not restricted to the G₁-phase but also included the S phase. These results support the idea that HLA class I molecules are important in the PHA-induced proliferation of human T-lymphocytes.     

Suppression of Interleukin-2 Receptor Expression on Mouse CD4+ T Cells by Bovine κ-Caseinoglycopeptide

Bovine K-caseinoglycopeptide (residues 106–169, CGP) completely inhibited the PHA-induced proliferation of mouse splenocytes when CGP was added simultaneously with PHA. The inhibitory effect, however, was reduced to about one-half when CGP was added after 24 h of cultivating the splenocytes with PHA or when anti-IL-lra antibody was added simultaneously with PHA and CGP. On the other hand, CGP bound to mouse CD4 ⁺ T cells but not to CD8⁺ T cells. CGP suppressed IL-2 receptor expression of the PHA-stimulated mouse CD4⁺ T cells.     

Immunosuppression of normal lymphoid cells by serum from mice undergoing chronic graft-vs-host disease.

Spleen cells from F1 mice undergoing chronic graft-vs-host (GVH) reaction, induced by injection of parental cells, were shown to be immunosuppressed since their in vitro responses to the mitogens concanavalin A (Con A) and bacterial lipopolysaccharide (LPS) were substantially lower than control animals. Serum, from mice undergoing GVH, when cultured in vitro with normal spleen cells was immunosuppressive. The proliferation response to Con A and allogeneic cells of normal syngeneic, allogeneic, and parental spleen cells was 90% suppressed when serum from mice undergoing chronic GVH was added in comparison to the addition of serum from untreated F1 mice. Similarly, the in vitro antibody response to a T-dependent antigen was impaired; however, the antibody response to a T-independent antigen was not impaired. These results indicate that T cell functions are more sensitive than are B cell functions to immunosuppressive factors in the serum of mice undergoing GVH.     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Suppression of Splenic Lymphocyte Proliferation by Eucommia ulmoides and Genipin

We investigated the modulation of innate and adaptive immune cell activation by Eucommia ulmoides Oliver extract (EUE) and its ingredient genipin. As an innate immunity indicator, the phagocytic activity of macrophages was determined by measuring engulfed, fluorescently labeled Escherichia coli. As a surrogate marker for the respective activation of cellular and humoral adaptive immunity, concanavalin A (Con A) and lipopolysaccharide (LPS) induction of primary splenocyte proliferation was assayed in in vitro and ex vivo systems. EUE and genipin suppressed the proliferation of primary splenic lymphocytes induced by Con A or LPS, but not macrophage phagocytosis. Oral administration of EUE and genipin to mice decreased splenic lymphocyte proliferation induced by Con A or LPS. These results revealed that E. ulmoides and genipin suppressed cellular and humoral adaptive immunity, and they suggest that E. ulmoides and genipin are promising candidates for immunosuppressive drugs that target diseases that involve excessive activation of adaptive immunity.     

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.     

Characterization of Cell Growth-Inhibitory Factor in Inflammatory Peritoneal Exudate Cells of Rats

We characterized the nature and reaction mode of the cell growth-inhibitory factor (here designated CGIF) from rat peritoneal exudate cells (PEC). The soluble fraction separated from the lysate of Enterococcus faecalis-induced 24 hr PEC completely inhibited Con A-induced thymocyte mitogenesis. Gel filtration chromatography showed that CGIF has a molecular weight of approximately 23–25 kDa. Isoelectric focusing with Rotofor indicates that the factor has an isoelectronic point of 5.8–6.4. CGIF was inactivated by treatment at 70 C, for 30 min or by tryptic digestion, but the activity was not destroyed by the reduction with dithiothreitol. As well as thymocyte proliferation, CGIF completely suppressed ³H-thymidine incorporation of splenocytes which were stimulated by either Con A or LPS, suggesting the factor is effective on both T and B cells. The acting point of the inhibitor appeared to be a later stage of the lymphocyte activation sequence, since it was still effective when added 28.5 hr after the addition of Con A. CGIF also reduced the viability of these cells when added with mitogens such as Con A or LPS. CGIF thus appears to be distinct from interleukin-1 receptor antagonist or transforming growth factor-β.     

Modulatory effects of garlic extracts on proliferation of T-lymphocytes in vitro stimulated with concanavalin A.

Different active components from the garlic (Allium sativum) possess immunomodulatory activity both in vitro and in vivo. However, mechanisms of their actions are not sufficiently elucidated. In this study we examined the effects of garlic aqueous extract (GAE) and garlic ethanolic extract (GEE), prepared from two different garlic powder samples, on proliferation of rat thymocytes and splenocytes in culture, stimulated with concanavalin A (Con A). It has been shown that the extracts from both samples significantly modulate lymphocyte proliferation, triggered by this potent T-cell mitogen, depending on the type and dilutions of extracts and concentrations of Con A. The extracts, alone, were not mitogenic for lymphocytes. Generally, higher concentrations of the extracts showed inhibitory effects, whereas lower concentrations significantly augmented proliferation of lymphocytes. The stimulatory effect of GAE was stronger using splenocytes and suboptimal concentrations of Con A as a consequence of increased interleukin 2 (IL-2) production as well as the expression of IL-2 receptor alpha (IL-2Ralpha). The relationship between these two phenomena was demonstrated using neutralizing anti-IL-2Ralpha monoclonal antibody. The inhibitory effect of GAE correlated with a decrease in IL-2 production, but was not followed by down-regulation of IL-2Ralpha expression. The addition of IL-2 almost completely abolished inhibition of lymphocyte proliferation in the presence of higher concentrations of GAE.