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The Journal of Immunology, 2001, 166: 72-80.
Copyright © 2001 by The American Association of Immunologists

Gangliosides GD1b, GT1b, and GQ1b Enhance IL-2 and IFN-{gamma} Production and Suppress IL-4 and IL-5 Production in Phytohemagglutinin-Stimulated Human T Cells1

Naoko Kanda2 and Shinichi Watanabe

Department of Dermatology, School of Medicine, Teikyo University, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gangliosides are sialic acid-containing glycolipids. We studied the in vitro effects of gangliosides on Th1 and Th2 cytokine production in PHA-stimulated human T cells. Gangliosides GD1b, GT1b, and GQ1b (each 100 nM) enhanced PHA-induced IL-2 secretion of peripheral blood T cells ~4-fold and enhanced that of IFN-{gamma} 3- to 4-fold compared with controls. These gangliosides decreased PHA-induced IL-4 secretion by 50–53% and that of IL-5 by 53–63% compared with controls, respectively. The other gangliosides did not alter the secretion of Th1 or Th2 cytokines. RT-PCR showed that GD1b, GT1b, and GQ1b enhanced PHA-induced IL-2 and IFN-{gamma} transcription and suppressed that of IL-4 and IL-5. Transient transfection assays of Jurkat T cells showed that GD1b, GT1b, and GQ1b enhanced PHA-induced IL-2 and IFN-{gamma} promoter activities but suppressed those of IL-4 and IL-5. The cAMP analogue dibutyryl cAMP and the cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine each reversed GD1b-, GT1b-, and GQ1b-induced stimulation of IL-2 and IFN-{gamma} production and inhibition of IL-4 and IL-5 production at the levels of proteins, transcription, and promoter activities. GD1b, GT1b, and GQ1b suppressed PHA-induced increase in cAMP level in T cells. These gangliosides suppressed PHA-stimulated adenylate cyclase activity in T cells. These results suggest that GD1b, GT1b, and GQ1b may enhance Th1 cytokine production while suppressing Th2 production by inhibiting adenylate cyclase activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Gangliosides are sialic acid-containing glycosphingolipids that are constituents of the plasma membranes of various cells (1). Some gangliosides are shed into the extracellular environment and exhibit physiologic functions (1). Exogenous gangliosides interact with plasma membranes and modulate transmembrane signaling pathways and thus regulate cell growth and differentiation (2). Especially, gangliosides regulate cAMP-related signaling pathways (3, 4, 5, 6). cAMP binds to protein kinase A (PKA)3 and up-regulates the activity of PKA to phosphorylate proteins at the serine/threonine position (7). Intracellular cAMP level is controlled by cAMP-synthesizing adenylate cyclase (AC) (3) and by cAMP-hydrolyzing cyclic nucleotide phosphodiesterase (PDE) (4). It is reported that gangliosides modulate the activities of AC, PDE, and PKA; mixed brain gangliosides enhanced AC and PDE activities from rat cerebral cortex (3, 4). Gangliosides GM1, GD1a, and GT1b inhibited the activity of the PKA catalytic subunit from porcine heart (5).4 Gangliosides also regulate cellular and humoral immune responses; gangliosides GM1, GD1a, and GD1b inhibited LPS-induced proliferation of murine splenocytes (8). GQ1b enhanced Ig production of human PBMC (9). GM2 and GM3 inhibited TNF-{alpha} and/or IL-10 production in Staphylococcus aureus Cowan strain I plus IL-2-activated human B cells (10, 11). It is plausible that some of these immunomodulatory effects by gangliosides may be manifested by regulating signal transduction pathways, including that related to cAMP.

Previous studies reported the regulatory effects of cAMP on cytokine production in murine and human T cells (12, 13, 14, 15, 16). cAMP suppresses the production of Th1 cytokines such as IL-2 or IFN-{gamma} that stimulate cellular immune responses (15, 16). In contrast, cAMP does not alter (12) or stimulate the production of Th2 cytokines such as IL-4 or IL-5 that promote humoral immune responses (14). These previous reports suggest that the modulation of cAMP signal may alter the T cell cytokine profile. It is thus possible that gangliosides may regulate Th1 and Th2 cytokine production via cAMP-related signaling pathways.

In this study, we examined the effects of individual gangliosides on the production of Th1 (IL-2 and IFN-{gamma}) and Th2 cytokines (IL-4 and IL-5) in PHA-stimulated human T cells. We have found that gangliosides GD1b, GT1b, and GQ1b selectively enhance Th1 cytokine production while suppressing Th2 production. We further examined the involvement of cAMP in the ganglioside-induced regulation of Th1/Th2 production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

PHA-P; a purified form of PHA from Phaseolus vulgaris; and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, and GQ1b were purchased from Sigma (St. Louis, MO). N-acetylneuraminic acid was obtained from Dextra Laboratories (Reading, U.K.). Ceramide was obtained from Research Biochemicals International (Natick, MA). Dibutyryl (Bt2) cAMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Calbiochem (La Jolla, CA).

Human T cells and T cell line

Blood was taken from five healthy Japanese volunteers (two men and three women age 43.6 ± 13.6 years, mean ± SD), who were informed of the objectives and methods of this study and consented to participate. PBMC were isolated by centrifugation over Ficoll-Paque (Pharmacia, Uppsala, Sweden) as described (17) and were allowed to adhere to plastic dishes for 1 h at 37°C. From the nonadherent cells, CD56- cells were isolated by negative selection using immunomagnetic beads (Dynal, Great Neck, NY) as described (18) and were incubated with neuraminidase-treated SRBC as described (19). From the rosette-forming cells, CD14- and CD19- cells were isolated by immunomagnetic negative selection and were used as T cells. This T cell population was >98% CD3+, and the contamination of CD14+, CD19+, or CD56+ cells was <2%.

Human Jurkat T cells were purchased from Dainippon Pharmaceutical (Osaka, Japan) and were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Measurement of cytokine secretion

T cells (2 x 105/200 µl/well) were cultured in triplicate in 96-well plates with or without 10 µg/ml PHA in the culture medium at 37°C in an atmosphere of 5% CO2 in air for 24 h. We used endotoxin-, hormone-, and serum-free medium and a 1:1 mixture of DMEM and Ham’s Nutrient Mixture F-12 (Sigma) supplemented with 2.5 mM L-glutamine (Life Technologies). The activity of IL-2, IFN-{gamma}, IL-4, and IL-5 in the culture supernatants was measured by ELISA kits (Biosource, Tokyo, Japan) according to the manufacturer’s instructions. The sensitivity of the assay for IL-2, IFN-{gamma}, IL-4, and IL-5 was 5, 4, 3, and 4 pg/ml, respectively.

RT-PCR

T cells were incubated under the conditions indicated, and total cellular RNA was extracted using mRNA purification kit (Pharmacia, Uppsala, Sweden) according to the manufacturer’s instructions. cDNA was made from RNA samples as described (20). PCR was performed using primer sets (Table IGo) in the thermal cycler programmed for 93°C for 1 min, 60°C for 1 min, and 72°C for 2 min for 35 cycles. The PCR products were analyzed by electrophoresis on 2.5% agarose gels and stained with ethidium bromide. The intensity of the bands for cytokine and {beta}-actin RT-PCR products was determined by densitometry (Hoefer Scientific Instruments, San Francisco, CA). Results are expressed for each cytokine product as the ratio relative to {beta}-actin product.


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  		Table I. Primers used for RT-PCR

 
Measurement of cAMP amount

T cells were cultured under the indicated conditions and were harvested and lysed with ethanol. The cell lysates were centrifuged, and the supernatants were dried under vacuum. The dried samples were dissolved in acetate buffer (pH 5.8), and cellular cAMP contents were measured with an ELISA kit from Amersham (Arlington Heights, IL) according to the manufacturer’s instructions. The sensitivity of the assay was 12 fmol/assay well. The cellular cAMP level was presented as pmol/106 cells.

Measurement of PDE activity

T cells were cultured under the indicated conditions and were lysed in the buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 15 mM benzamidine, and 3.75 mM 2-ME. PDE activity of the cell lysates was assayed as described (21, 22) using 1 µM 2,8-[3H]cAMP (30 Ci/mmol) (Amersham) as a substrate. The assays were performed in 40 mM Tris-HCl (final pH 8.0) and 10 mM MgCl2 at 37°C for 10 min, and PDE activity was presented as pmol cAMP hydrolyzed/min/mg protein.

Measurement of AC activity

The T cell lysate was centrifuged at 23,600 x g for 10 min. The pellet was used as a particulate fraction for AC assays as described (23, 24). The AC activity of the fraction was measured at 37°C for 10 min in 20 mM Tris-HCl (pH 7.4), 1 mM [{alpha}-32P]ATP (30 Ci/mmol) (Amersham), 1 mM [3H]cAMP, 1 mM IBMX, 5 mM MgCl2, 0.2 mM EGTA, 20 mM creatine phosphate, and 100 U/ml creatine phosphokinase. AC activity was presented as pmol cAMP formed/min/mg protein.

Plasmids and transfection

pCAT3-basic vector carrying two SV40 poly(A) signals, one downstream of the chloramphenicol acetyltransferase (CAT) reporter gene and the other upstream of the multicloning site, was purchased from Promega (Madison, WI). The plasmid IL-2-CAT, which contains human IL-2 promoter (-541 to +42 bp relative to the transcriptional start site) was generated by PCR using human genomic DNA (Clontech, Heidelberg, Germany) and primers based on the reported sequence (25) and was cloned into the NheI-BglII site of the pCAT3-basic vector. The plasmids IFN-{gamma}-, IL-4-, and IL-5-CAT, containing the promoters of IFN-{gamma} (-337 to +64 bp), IL-4 (-418 to +50 bp), and IL-5 (-511 to +4 bp), respectively, were generated as described (26, 27, 28). The entire cloned regions were sequenced by the chain termination method and found to be identical with the reported genomic sequences (25, 29, 30, 31). Transfection of Jurkat cells was conducted by the DEAE-dextran method as described (14). Cells (107) were incubated with 10 µg/ml DNA and 350 µg/ml DEAE-dextran (Pharmacia, Uppsala, Sweden) in Tris-buffered saline for 30 min at room temperature. To decrease variations in transfection efficiency, cells were transfected in single batches, which were then separated into different drug treatment groups. Cells were washed with Tris-buffered saline and incubated with DMEM/F12 medium. After 24 h, cells were treated with different combinations of stimuli for 16 h, and then the cells were harvested and lysed by three freeze/thaw cycles. The cell lysate was centrifuged, and supernatant was assayed for CAT expression by CAT-ELISA (Roche Diagnostics, Tokyo, Japan) according to the manufacturer’s instructions. The total protein amount was measured by a Bradford microassay procedure (Bio-Rad, Hercules, CA). The expression of various CAT plasmids was presented as pg CAT enzyme synthesized/µg total protein. pCAT3-control vector (Promega) containing SV40 early promoter and enhancer sequences was used as positive control, and promoterless pCAT3-basic vector was used as negative control.

Assays of PKA

The T cell lysates were centrifuged at 23,600 x g for 10 min. The supernatant was assayed for the activity of PKA by an ELISA kit (Medical and Biological Laboratories, Nagoya, Japan), using synthetic peptide-precoated microtiter plates and a mAb recognizing phosphorylated form of the peptide. The PKA activity was expressed as OD at 492 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of various gangliosides on PHA-induced IL-2, IFN-{gamma}, IL-4, and IL-5 secretion

First, various gangliosides were added to the T cell culture with PHA, and their effects on the secretion of Th1 and Th2 cytokines were examined. GD1b, GT1b, and GQ1b enhanced PHA-induced IL-2 and IFN-{gamma} secretion, while they reduced that of IL-4 and IL-5 (Fig. 1Go). Both GD1b-induced stimulatory effects on Th1 and inhibitory effects on Th2 secretion were detected at 1 nM, increased dose dependently, and were maximized at 100 nM. This also appeared to be an optimal concentration for GT1b and GQ1b; 100 nM GD1b, GT1b, and GQ1b enhanced PHA-induced IL-2 secretion 4.02-, 3.98-, and 3.97-fold, and that of IFN-{gamma} 4.45-, 3.75-, and 3.25-fold as compared with controls, respectively (Fig. 1Go). In contrast, 100 nM GD1b, GT1b, and GQ1b reduced PHA-induced IL-4 secretion by 50, 52.5, and 53% and that of IL-5 by 52.8, 57.8, and 63.3% as compared with controls, respectively (Fig. 1Go). The other gangliosides, GM1, GM2, GM3, GD1a, GD2, and GD3, did not alter the PHA-induced secretion of Th1 or Th2 cytokines. N-acetylneuraminic acid or ceramide did not alter the PHA-induced Th1 or Th2 secretion, either (data not shown). GD1b, GT1b, and GQ1b did not alter spontaneous Th1 and Th2 secretion without PHA; IL-2 secretion in T cells cultured with medium alone, GD1b, GT1b, or GQ1b was 20 ± 12, 21 ± 11, 19 ± 12, and 18 ± 13 pg/ml, respectively (mean ± SEM; n = 5). The spontaneous secretion of IFN-{gamma}, IL-4, or IL-5 was at a less-than-detectable level in both the presence and absence of these gangliosides. The other gangliosides, N-acetylneuraminic acid, or ceramide did not alter spontaneous Th1 or Th2 secretion either (data not shown). Thus gangliosides GD1b, GT1b, and GQ1b enhance Th1 secretion, while they suppress Th2 secretion in PHA-stimulated T cells. We then examined whether these gangliosides may also regulate PHA-induced expression of mRNA for Th1 and Th2 cytokines.



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  		FIGURE 1. Dose dependency for the effects of various gangliosides on PHA-induced secretion of Th1 and Th2 cytokines. Human peripheral blood T cells were cultured with 10 µg/ml PHA in the presence or absence of indicated doses of gangliosides. After 24 h, the culture supernatants were assayed for IL-2, IFN-{gamma}, IL-4, and IL-5 by ELISA. Values are means ± SD of triplicate cultures. The data represent five separate experiments using T cells from five different donors. *, p < 0.05 vs cultures with PHA alone by one-way ANOVA with Dunnett’s multiple-comparison test.

 
The effects of GD1b, GT1b, and GQ1b on PHA-induced Th1 and Th2 mRNA expression

As analyzed by RT-PCR, GD1b, GT1b, and GQ1b enhanced PHA-induced IL-2 and IFN-{gamma} mRNA expression while they reduced that of IL-4 and IL-5 at 3 h of culture of peripheral blood T cells (Fig. 2Go). These gangliosides did not alter Th1 or Th2 mRNA expression in the absence of PHA (data not shown). The other gangliosides, N-acetylneuraminic acid, or ceramide did not alter the PHA-induced Th1 or Th2 mRNA expression (data not shown). Thus the effects of GD1b, GT1b, and GQ1b on Th1 and Th2 mRNA expression closely correlated with those on protein secretion, suggesting the pretranslational regulation by these gangliosides. We then examined whether these gangliosides may exert their action at the transcriptional level by analyzing the effects of the gangliosides on the activities of promoters for Th1 and Th2 cytokines.



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  		FIGURE 2. The effects of GD1b, GT1b, and GQ1b on PHA-induced expression of mRNA for Th1 and Th2 cytokines. Human peripheral blood T cells were cultured with or without 10 µg/ml PHA in the presence or absence of indicated gangliosides (each 100 nM) for 3 h, and RNA was extracted. RT-PCR products were analyzed by electrophoresis (A), and the intensity of the products was determined by densitometry (B). The data represent five separate experiments using T cells from five different donors.

 
The effects of GD1b, GT1b, and GQ1b on Th1/Th2 promoter activities

Human Jurkat T cells were transiently transfected with plasmids containing human IL-2, IFN-{gamma}, IL-4, or IL-5 promoters driving CAT reporter gene and stimulated with PHA in the presence or absence of gangliosides. The promoter activity was assessed by the expression of CAT enzyme. The attempt to transfect fresh peripheral blood T cells was unsuccessful (data not shown). As shown in Table IIGo, GD1b, GT1b, or GQ1b did not alter the basal activity of each promoter without PHA induction. However, all of these gangliosides enhanced the PHA-induced IL-2 and IFN-{gamma} promoter activities. In contrast, these gangliosides reduced the PHA-induced IL-4 and IL-5 promoter activities. The other gangliosides, ceramide, or N-acetylneuraminic acid did not alter Th1 or Th2 promoter activity (data not shown). These results in transfection assays are consistent with those in mRNA expression and protein secretion for Th1/Th2 cytokines. It is thus suggested that GD1b, GT1b, and GQ1b may regulate Th1 and Th2 production at the transcriptional level, although posttranscriptional regulation is also implicated.


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  		Table II. The effects of gangliosides on the activities of promoters for Th1 and Th2 cytokines1

 
It is known that gangliosides modulate a variety of intracellular signals (2). The differential effects of GD1b, GT1b, and GQ1b on Th1 and Th2 production indicate the involvement of a certain signal(s) that differentially regulates Th1 and Th2 production. cAMP is one of these; PKA, cAMP-dependent protein kinase, may suppress Th1 cytokine production while it may not alter or enhance Th2 production (12, 13, 14). It is known that PHA transiently activates AC and elevates cAMP level and thus activates PKA (32). It is thus plausible that GD1b, GT1b, and GQ1b may suppress the PHA-induced cAMP increase and thus enhance Th1 while they suppress Th2 production. We thus examined whether the elevation of cAMP level may counteract the differential effects of gangliosides on Th1 and Th2 cytokine production.

cAMP-induced reversal of GD1b, GT1b, and GQ1b-induced effects on Th1 and Th2 production

cAMP-elevating agents were added to the culture with GD1b in the presence of PHA, and their influence was tested on the effect of each ganglioside. The agents were the AC stimulator forskolin, the cAMP analogue Bt2cAMP, and the PDE inhibitor IBMX. As shown in Fig. 3Go, these agents were added at low concentrations that did not influence Th1 or Th2 production induced by PHA alone. The cAMP-elevating agents counteracted the effects of GD1b both on Th1 and on Th2 protein secretion; the agents completely blocked GD1b-induced stimulation of IL-2 and IFN-{gamma} secretion. The agents also reversed GD1b-induced reduction of IL-4 and IL-5 secretion. In contrast, cGMP analogue Bt2cGMP did not reverse the effects of GD1b on Th1 and Th2 cytokine secretion, indicating that the reversal is specific to cAMP. The cAMP-elevating agents also counteracted the effects of GT1b and GQ1b on Th1 and Th2 cytokine secretion in PHA-stimulated T cells (data not shown). The cAMP-elevating agents also reversed the effects of these gangliosides on Th1 and Th2 mRNA expression as examined by RT-PCR (data not shown).



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  		FIGURE 3. cAMP-mediated reversal of the GD1b effects on PHA-induced secretion of Th1 and Th2 cytokines. Peripheral blood T cells from five different donors were preincubated with or without 50 µM Bt2cAMP, 1 µM forskolin (FSK), 50 µM IBMX, or 50 µM Bt2cGMP for 30 min before the addition of 10 µg/ml PHA with or without 100 nM GD1b. After 24 h, cytokine secretion was analyzed by ELISA. Values are means ± SEM (n = 5). *, p < 0.05 vs cultures with PHA alone; {dagger}, p < 0.05 vs cultures with PHA plus GD1b, by one-way ANOVA with Scheffé’s multiple comparison test.

 
We then examined whether the increase of cAMP may also reverse the effects of GD1b on Th1 and Th2 promoter activities. As shown in Fig. 4Go, Bt2cAMP counteracted the stimulation by GD1b on PHA-induced IL-2 and IFN-{gamma} promoter activities in Jurkat T cells. Bt2cAMP also reversed the suppression by GD1b on PHA-induced IL-4 and IL-5 promoter activities. In contrast, Bt2cGMP did not reverse the effects of GD1b on Th1 and Th2 promoter activities, indicating the specific effect of cAMP. Bt2cAMP also reversed the effects of GT1b and GQ1b on Th1 and Th2 promoter activities (data not shown). Similar results were obtained using the other cAMP-elevating agents forskolin (1 µM) and IBMX (50 µM) (data not shown). Thus the increase of cAMP reversed the effects of gangliosides at the transcriptional level. It is thus indicated that the effects of GD1b, GT1b, and GQ1b may be attributable to the reduction of cAMP level; these gangliosides may suppress the PHA-induced increase of cAMP and thus prevent the cAMP-induced inhibition of Th1 production and cAMP-induced stimulation of Th2 production. We then examined whether these gangliosides may reduce cAMP level in T cells.



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  		FIGURE 4. cAMP-mediated reversal of the GD1b effects on PHA-induced promoter activities for Th1 and Th2 cytokines. Jurkat T cells were transfected with IL-2, IFN-{gamma}, IL-4, or IL-5 promoter-CAT reporter constructs. After transfection, cells were cultured with medium for 16 h and then preincubated with or without Bt2cAMP or Bt2cGMP (each 50 µM) for 30 min before the addition of 10 µg/ml PHA with or without GD1b 100 nM. After 24 h, cell extracts were assayed for CAT expression. Values are means ± SD of triplicate assays. *, p < 0.05 vs cultures with PHA alone; {dagger}, p < 0.05 vs cultures with PHA plus GD1b, by one-way ANOVA with Scheffé’s multiple-comparison test. The CAT expression by pCAT3 control vector without stimuli was 162 ± 14 pg CAT/µg protein, and that by pCAT3 basic vector was at a less-than-detectable level. The data are representative of five separate experiments.

 
The effects of gangliosides on cAMP level and PKA activity in PHA-stimulated T cells

As shown in Table IIIGo, PHA increased cAMP amount of T cells 4.5-fold above the basal level, and the increase was blocked by GD1b, GT1b, and GQ1b; however, it was not blocked by the other gangliosides, N-acetylneuraminic acid, or ceramide (data not shown). The basal cAMP amount in the absence of PHA appeared to be slightly reduced by GD1b, GT1b, and GQ1b; however, the differences from controls were not significant. PHA enhanced PKA activity, which correlated with the PHA-induced increase of cAMP. GD1b, GT1b, and GQ1b blocked the PHA-induced activation of PKA, while the other gangliosides, N-acetylneuraminic acid, and ceramide (data not shown) were ineffective. The basal activity of PKA in the absence of PHA seemed to be slightly suppressed by GD1b, GT1b, and GQ1b; however, the differences from controls were not significant. The direct addition of each ganglioside to the reaction mixture did not alter the activity of PKA (data not shown), indicating that GD1b, GT1b, and GQ1b may not directly act on PKA but may suppress its activity by decreasing cAMP level. Because the decrease of cAMP level can be mediated by the inhibition of AC and/or stimulation of PDE, we then examined whether GD1b, GT1b, and GQ1b may alter AC and/or PDE activity.


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  		Table III. The effects of gangliosides on cAMP level and protein kinase A activity in T cells1

 
The effects of gangliosides on AC and PDE activities in PHA-stimulated T cells

As shown in Table IVGo, PHA increased AC activity of T cells 4-fold above the basal level, and the increase was suppressed by GD1b, GT1b, and GQ1b. GD1b, GT1b, and GQ1b also significantly reduced the basal AC activity in the absence of PHA. In contrast, the other gangliosides, N-acetylneuraminic acid, and ceramide (data not shown) did not alter basal or PHA-stimulated AC activity. PHA also enhanced PDE activity of T cells slightly (1.68-fold) above the basal level, and none of the gangliosides altered basal or PHA-stimulated PDE activity. Because it is reported that PDE activity is increased at later time points after PHA stimulus (on the order of hours or days) (22), we examined PDE activity over a culture period ranging from 30 min to 72 h, when PDE activity in PHA-stimulated T cells increased maximally 3- to 4-fold above the basal level. However, over this period, we could not detect significant effects of gangliosides on basal or PHA-stimulated PDE activity (data not shown). These results suggest that GD1b, GT1b, and GQ1b may suppress AC activity without altering that of PDE and thus may reduce cAMP level in PHA-stimulated T cells. When each ganglioside was directly added to the reaction mixture, GD1b, GT1b, and GQ1b dose-dependently inhibited AC activity of particulate fraction from T cells (Fig. 5Go). The optimal ganglioside concentration for AC inhibition was 100 nM, which was equal to that for the effects on Th1 and Th2 production. The other gangliosides were ineffective. These results suggest that GD1b, GT1b, and GQ1b may directly act on AC associated with T cell membranes and suppress its activity. The ganglioside-induced inhibition of AC may prevent the cAMP-induced inhibition of Th1 and enhancement of Th2 cytokine production, which may result in the stimulation of Th1 and inhibition of Th2 production.


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  		Table IV. The effects of gangliosides on AC and PDE activities of T cells1

 


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  		FIGURE 5. Dose dependency for the effects of various gangliosides on AC activity of particulate fraction from T cells. The particulate fraction of peripheral blood T cells was assayed for AC activity in the presence or absence of indicated doses of gangliosides. Values are means ± SD of triplicate assays. The data represent five separate experiments using T cells from five different donors. *, p < 0.05 vs cultures with PHA alone by one-way ANOVA with Dunnett’s multiple-comparison test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, gangliosides GD1b, GT1b, and GQ1b enhanced IL-2 and IFN-{gamma} production, while they reduced IL-4 and IL-5 production in PHA-stimulated human T cells, although these gangliosides alone did not induce the production of these cytokines. The differential effects of gangliosides were mediated by the inhibition of PHA-induced AC activation. On the T cell membranes, PHA binds to TCR/CD3 complexes (33). Although TCR/CD3 complexes do not directly couple to AC, the binding of PHA to the complexes triggers the activation of phospholipase C, which produces second messengers, the activation and membrane translocation of protein kinase C, and intracellular Ca2+ mobilization (34, 35). It is reported that either or both of the second messengers may activate AC; protein kinase C activates AC by phosphorylation, while Ca2+ forms a complex with intracellular calmodulin, and the Ca2+/calmodulin complex binds to and activates AC (34). PHA also binds to CD2 (33, 34), which induces the activation of AC possibly via intracellular Ca2+ signal (35, 36). Thus PHA binding to T cells indirectly activates AC and generates early cAMP signal (34). However, the cAMP signal suppresses Th1 gene transcription via PKA (15, 37, 38, 39, 40). PKA-phosphorylated cAMP-responsive element binding protein (CREB) and/or activating transcription factor-1 competitively inhibits the binding of AP-1 to the IFN-{gamma} promoter and thus suppresses the promoter activity (37, 38). PKA-mediated phosphorylation also alters the composition of NF-AT or AP-1 complexes and thus suppresses their function to activate IL-2 gene transcription (40). In contrast, for the Th2 cytokine production, stimulatory effects of cAMP are reported, although some conflicting data are also seen; cAMP up-regulated IL-4 and IL-5 transcription in Con A-primed and ionophore plus PMA-restimulated murine CD4+ T cells (13). cAMP activated IL-5 promoter in murine thymoma EL-4 cells (14) possibly by altering the composition of NF-AT-related complexes via PKA (40). In contrast, cAMP inhibited Con A-induced or anti-CD3 plus anti-CD28-induced IL-4 transcription in human peripheral blood T cells (41). Thus the regulatory effects of cAMP on Th2 cytokine production may vary depending on T cell sources and/or applied stimuli (42). In our present system using PHA-stimulated human T cells, cAMP enhanced IL-4 and IL-5 production. In contrast, cAMP blocked the GD1b-, GT1b-, and GQ1b-induced enhancement of IL-2 and IFN-{gamma} production in our present system. These gangliosides may thus switch T cell phenotype toward Th1 by reducing cAMP level through the inhibition of AC activity.

Most ACs are associated with plasma membranes (43). Transmembrane receptors for certain hormones, neurotransmitters, and other stimuli are coupled to AC via guanine nucleotide-binding proteins (G-proteins) (43). Gangliosides are known to modulate AC activity by several different mechanisms; gangliosides may alter 1) the ligand binding activity of the receptor, 2) the linkage between receptors and G-proteins, 3) the interaction between G-proteins and AC, and 4) the activity of AC catalytic domains (3, 44, 45, 46, 47, 48). The effects of gangliosides on AC may vary depending on ganglioside species, ganglioside concentrations, target cell types, and receptor classes (44). Because the putative PHA receptors, TCR/CD3 complex and/or CD2, do not directly couple to AC (34), it is most probable that gangliosides may directly inhibit the activity of AC catalytic domains. The inhibitory effects of GD1b, GT1b, and GQ1b occurred in almost the same magnitudes and at the submicromolar level, while the other gangliosides were ineffective. GD1b, GT1b, and GQ1b commonly contain the structure NeuAc(2->8)NeuAc attached to the internal galactose (49), indicating that this structure may be specifically required for the interaction with the AC catalyst. Alternatively, incorporation of these gangliosides into plasma membranes may decrease the fluidity of the lipid microenvironment surrounding AC, which may lead to the decrease of AC activity. The close relationship between lipid fluidity and AC activity is reported (43, 46, 50).

Partington and Daly (3) reported that mixed brain gangliosides increased basal AC activity of rat cerebral cortex membranes. Their results were in contrast with ours. The discrepancy may be, first, due to the difference in ganglioside concentrations; they used 50 µM mixed gangliosides, a concentration much higher than the submicromolar level in our study. The second reason for the discrepancy may reside in the difference in the AC subtypes; AC is classified into eight different subtypes mainly on the basis of the sequence homology, and all eight types have been isolated from the brain while only types 6 and 7 have been detected in cells of lymphocyte lineage (43). Because the activity of each AC subtype is distinctly regulated by a variety of agents such as Ca2+ or protein kinases (43), the up- or down-regulation by gangliosides may also vary depending on the AC subtype. AC subtypes in T cells may be susceptible to the inhibition by GD1b, GT1b, and GQ1b, while the brain may contain the different AC subtypes that may be stimulated by these three and/or the other gangliosides. Thus the ganglioside-inhibitable AC subtype(s) on T cell membranes should be identified in further studies. Dacremont et al. (45) also reported that gangliosides inhibited basal, thyrotropin-induced, and sodium fluoride-induced AC activity of human thyroid membranes, suggesting that gangliosides may directly inhibit the activity of AC catalyst. In their study, the order of the inhibitory effect on AC was GD3 > GD1b = GT1b > GD1a. Their results are mostly consistent with ours; however, extremely high concentrations (>100 µM) of gangliosides were used in their study, and thus the physiological importance is unknown.

In this study, gangliosides did not alter the PDE activity of T cells either in the presence or absence of PHA. However, several studies reported the stimulatory effects of gangliosides on the activity of PDE from brain; mixed brain gangliosides at micromolar levels enhanced the activity of PDE from rat cerebral cortex (14). GT1b, GD1a, and GM1 at nanomolar levels stimulated the activity of PDE from bovine brain (5). The results of these authors thus conflict with ours, which may be due to the difference in PDE subtypes between brain and lymphocytes. Nine different PDE subtypes (PDE 1–9) have been identified, and the PDE typing varies with cell types (51). We used cAMP as a substrate for T cell-derived PDE, whereas the previous studies used cGMP for brain-derived PDE, suggesting that gangliosides may stimulate brain-localized PDE subtypes hydrolyzing cGMP, possibly type 1, 2, 5, 6, or 9 (51). In contrast, PHA-stimulated T cells contain PDE 1, 3, 4, and 7, among which types 3 and 4 are main components and can hydrolyze cAMP but cannot hydrolyze cGMP (52). It is thus conceivable that certain gangliosides may activate PDE subtypes hydrolyzing cGMP but may not activate PDE 3 and 4. Thus, the effects of individual gangliosides on each PDE subtype should be further examined.

GD1b, GT1b, and GQ1b did not directly suppress PKA activity when added to the reaction mixture. These gangliosides may indirectly reduce PKA activity by decreasing cAMP level and may not directly act on PKA, at least at a nanomolar level. However, several papers reported the direct inhibitory effects of gangliosides on PKA; GM1, GD1a, and GT1b suppressed the activity of PKA catalytic subunit in the absence of cAMP (5). GM1 also reduced the activity of PKA holoenzyme in the presence of cAMP (6). However, these direct inhibitory effects were manifested at extremely high concentrations (~100 µM) and thus may only occur in ganglioside-enriched environments such as the brain.

GD1b, GT1b, and GQ1b at nanomolar levels enhanced Th1 and suppressed Th2 cytokine production in PHA-stimulated human T cells. Our present results are opposite those shown by Irani et al. (53), who reported that bovine brain gangliosides blocked IL-2 and IFN-{gamma} transcription, while they did not inhibit that of IL-4 and IL-10 in PMA plus ionophore-stimulated murine T cells. However, Irani et al. (53) examined the effect of mixed brain gangliosides, not individual species, at extremely high concentrations, 100–200 µg/ml (~40–80 µM). Their results may thus reflect the local T cell responses within the brain. In contrast, our results may reflect the systemic immune responses, because the serum levels of GD1b, GT1b, and GQ1b are close to the in vitro optimal concentration (100 nM) in the present study; the serum concentrations of GD1b and GT1b are 200–300 and 400–500 nM, respectively, and that of GQ1b is <200 nM (54). It is thus suggested that these gangliosides may systemically regulate in vivo Th1 and Th2 cytokine production. It is also indicated that these gangliosides may be involved in the development of diseases with Th1/Th2 imbalance. Rheumatoid arthritis, multiple sclerosis, and psoriasis are inflammatory diseases characterized by Th1-skewed immunity; T cells from patients with these diseases predominantly produce Th1-type cytokines, such as IFN-{gamma}, over Th2 (55, 56, 57), indicating the increase of GD1b, GT1b, or GQ1b in the patients’ sera. In contrast, T cells from patients with atopic dermatitis or asthma produce abnormally high amounts of Th2 cytokines in response to allergens or mitogens while Th1 responses are suppressed (58, 59), indicating the decrease of GD1b, GT1b, or GQ1b in the patients’ sera. It is reported that total serum ganglioside concentration is increased in multiple sclerosis patients by 34% compared with that in healthy donors and that GT1b is one of the main components (60). However, there are no reports showing the increase or decrease of GD1b, GT1b, or GQ1b in the other diseases with Th1/Th2 imbalance. Thus, we cannot directly prove that the abnormal concentrations of these gangliosides cause Th1/Th2 imbalance. However, these gangliosides can be used therapeutically for the atopic diseases with Th2-polarized immunity. GD1b, GT1b, and GQ1b may adjust the imbalance of Th1/Th2 in the atopic patients by promoting Th1 and inhibiting Th2 cytokine production. We are now studying the effects of these gangliosides on T cells from atopic donors.


   Acknowledgments
 
We thank Dr. Tomoo Tanase (Takara Gene Analysis Center) for the synthesis of pIL-2-CAT and pIFN-{gamma}-CAT.


   Footnotes
 
1 This work was supported by a grant from the Japanese Ministry of Education (10770389). Back

2 Address correspondence and reprint requests to Dr. Naoko Kanda, Department of Dermatology, Teikyo University, School of Medicine, 11-1, Kaga-2, Itabashi-Ku, Tokyo 173-8605, Japan. Back

3 Abbreviations used in this paper: PKA, protein kinase A; AC, adenylate cyclase; PDE, cyclic nucleotide phosphodiesterase; Bt2, dibutyryl; IBMX, 3-isobutyl-1-methylxanthine; CAT, chloramphenicol acetyltransferase; CREB, cAMP response element binding protein; G-protein, guanine nucleotide-binding protein. Back

4 The nomenclature for gangliosides used in this article follows the system of Svennerholm (49 ). Back

Received for publication August 9, 2000. Accepted for publication September 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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