Inhibition of mouse and rat lymphoproliferation by gangliosides

Abstract

Our previous study have demonstrated that Glycosphingolipids (GSLs) have an immunosuppressive effect on murine lymphoproliferation and IL-2 production. In the present study we examined the effect of a pool of Gangliosides (Gang) on spleen lymphocyte proliferation from either isogeneic strains of Wistar rats or BALB/c mice. Two hundred-fifty grams adult female isogeneic Wistar rats and 8-week-old BALB/c mice were used. The animals were sacrificed and the spleen harvested aseptically for cellular assays. Spleen cells suspensions were obtained by homogenization in RPMI 1640 with a loose tissue grinder. After washing, the cells were suspended in RPMI 1640 supplemented. Cell viability was measured by Trypan blue exclusion. Cells were cultured in triplicate using increasing concentrations of Gang (1; 2; 5; 10; 15; 20 mug/well) and in the presence of Concanavalin A. The cells were incubated for 48 hours and were pulsed with [³H] thymidine 18 hours prior to harvesting on glass fiber paper for counting in a beta-counter. Data were presented as rate of inhibition, as previously described. At concentrations 1 and 2 mug/well, Gang stimulated lymphoproliferation (30% and 50%, rats and mice respectively), while at concentration from 5 to 20 mug/well an increasing inhibition was observed for spleen cells from both mouse and rat (from 40% up to 80%). In preliminary studies we observed inhibition of mixed lymphocyte reaction on spleen cells from rats treated with Gang for 10 days (data not shown). Our data suggest that Gang may be investigated as a immunosuppressive drug in organ transplantation.

Gangliosides; Lymphoproliferation; Immunomodulation; Rats

INHIBITION OF MOUSE AND RAT LYMPHOPROLIFERATION BY GANGLIOSIDES

MONTERO EFS ¹,

CASTRO LC ²,

BARBIERI CL ³,

TAHA MO ⁴,

NIGRO AJT ⁵

Montero EFS, Castro LC, Barbieri CL, Taha MO, Nigro AJT. Inhibition of mouse and rat lymphoproliferation by gangliosides. Acta Cir Bras 2000; 15(Suppl 1):4-7.

ABSTRACT: Our previous study have demonstrated that Glycosphingolipids (GSLs) have an immunosuppressive effect on murine lymphoproliferation and IL-2 production. In the present study we examined the effect of a pool of Gangliosides (Gang) on spleen lymphocyte proliferation from either isogeneic strains of Wistar rats or BALB/c mice. Two hundred-fifty grams adult female isogeneic Wistar rats and 8-week-old BALB/c mice were used. The animals were sacrificed and the spleen harvested aseptically for cellular assays. Spleen cells suspensions were obtained by homogenization in RPMI 1640 with a loose tissue grinder. After washing, the cells were suspended in RPMI 1640 supplemented. Cell viability was measured by Trypan blue exclusion. Cells were cultured in triplicate using increasing concentrations of Gang (1; 2; 5; 10; 15; 20 mg/well) and in the presence of Concanavalin A. The cells were incubated for 48 hours and were pulsed with [³H] thymidine 18 hours prior to harvesting on glass fiber paper for counting in a b-counter. Data were presented as rate of inhibition, as previously described. At concentrations 1 and 2 mg/well, Gang stimulated lymphoproliferation (30% and 50%, rats and mice respectively), while at concentration from 5 to 20 mg/well an increasing inhibition was observed for spleen cells from both mouse and rat (from 40% up to 80%). In preliminary studies we observed inhibition of mixed lymphocyte reaction on spleen cells from rats treated with Gang for 10 days (data not shown). Our data suggest that Gang may be investigated as a immunosuppressive drug in organ transplantation.

SUBJECT HEADINGS: Gangliosides.Lymphoproliferation.Immunomodulation.Rats

INTRODUCTION

Our previous study has demonstrated that glycosphingolipids (GSLs) exert an immunomodulatory effect on murine lymphoproliferation and IL-2 production¹. GSLs obtained from various organs induced different levels of inhibition on "in vitro" lymphocyte proliferation in a dose-dependent pattern. GSLs extracted from testis had showed more immunosuppressive effect than that induced by GSLs obtained from brain. At that time, we were not able to show any correlation between immunoprivileged organs and GSLs potency of inhibition. GSLs are found only in the outer half of the lipidic bilayer of the cell membrane, while their carbohydrate moieties are exposed at the cell surface suggesting their possible role in interactions of the cell with its surroundings^2,3. To verify if some components are more important than others, in the present work, we decide to examine the effect of one type of GSLs, gangliosides (Gang) extracted from brain on spleen lymphocytes proliferation from either isogeneic strains of Wistar rats or BALB/c mice, in vitro.

METHODS

Two hundred-fifty grams adult isogeneic Wistar rats and 8-week-old BALB/c mice were used. The animals were sacrificed in Ethylic Ether saturated chamber and the spleen harvested aseptically by a full-length abdominal skin and muscle incision. A mixture of gangliosides from bovine brain, containing GM1 (21%), GD1a (40%), GD1b (16%), and GT1b (19%), (Sinaxial ^R) were subjected to HPTLC in a solvent system (chloroform/ methanol/ CaCl₂ 0.02%, 60/40/9, v/v/v).

Proliferation Assays: Spleen cell suspensions were obtained by homogenization in RPMI 1640 with a loose tissue grinder. After washing, the cells were suspended in RPMI 1640 supplemented with 0.02 mM sodium bicarbonate, 5% normal mice serum, 100 U/mL penicilin, 100 mg/mL streptomycin sulphate, 1.5 mM L-glutamine, 5x10^-5 mM -mercaptoethanol. Cell viability was measured by Trypan blue exclusion. Cells were cultured in triplicate using 3x10^ cells/well in a 96-well plates adding increasing concentration of Gang (1; 2; 5; 10; 15; 20 mg/well) and fixed concentration of Concanavalin A (2.5 mg/mL). The cells were incubated at 37^C in 5% CO₂ for 48 hours. Cells were pulsed with 1 mCi of [³H] thymidine ([³H]TdR-86Ci/mmol) per well 18 hours before they were harvested on glass fiber paper. [³H]TdR uptake was measured in a b-scintilator counting. The percentage of proliferative modulation was calculated as previously described ⁴.

RESULTS

Gang stimulated lymphoproliferation at concentrations of 1 and 2 mg/well (30% and 50%, from rats and mice, respectively), while at concentration from 5 to 20 mg/well an increasing inhibition was observed in spleen cells from both mouse and rat (from 40% up to 80%). (table I, fig. 1)

DISCUSSION

These results showed a non-specific immunomodulation on mouse and rat lymphoproliferation. Comparison of the immunomodulatory effect presented by neutral GSLs and Gang showed a significative difference. GSLs inhibited lymphocyte proliferation in a dose-dependent manner, while Gang stimulated lymphoproliferation at lower doses and presented inhibitory effect at higher doses. In our previous work, we observed differences on lymphoproliferation inhibition in the presence of GSLs extracted from testis and brain¹. One hypothesis which may explain this difference is relative to the composition of glycosphingolipids present in those organs, because a high concentration of gangliosides was found in the brain compared to testis. Data from De Ruysscher et al corroborate this hypothesis showing that asialo GM1-positive cells were able to prevent Graft-Versus-Host disease when present in the host⁵. It would be interesting to verify the components of glycolipids obtained from different organs on lymphocyte proliferation, because when we used GSLs from brain there was only inhibition. Otherwise, when it was used just gangliosides it occurred stimulation of lymphoproliferation using low doses. Maybe, some interaction among the components of GSLs can alter their properties. In preliminary studies we observed inhibition of mixed lymphocyte reaction on spleen cells from rats treated "in vivo" with Gang for 10 days (data not shown). Taken together, these data open perspectives to test Gang as an immunosuppressive drug in organ transplantation.

Our data showed that gangliosides present an immunomodulatory effect on murine lymphoproliferation. Although, those glycoconjugates were stimulating at low doses, at higher doses were able to inhibit the lymphoproliferation.

• 1. Montero EFS; Barbieri CL; Giorgio S; Garcez-Silva MH; Sato H; Goldenberg S; Straus AH; Takahashi HK; Koh IHJ. Immunomodulatory effects of Glycosphingolipids on lymphoproliferation and IL-2 production in rodents. Transplant Proc 1994;26:1597-8.
• 2. Hakomori S. Bifunctional role of Glycosphingolipids. J Biol Chem 1990;265:18713-6.
• 3. Marcus DM.. A review of immunogenic and immunomodulatory properties of glycosphingolipids. Mol Immunol 1984;21:1083-91.
• 4. Giorgio S; Jasiulionis MG; Straus AH; Takahashi HK; Barbieri CL. Inhibition of mouse proliferative response by Glycosphingolipids from Leishmania (L.) amazonensis. Exp Parasitol 1992;75:119-25.

Trabalho realizado na Disciplina de Técnica Operatória e Cirurgia Experimental e de Parasitologia da Universidade Federal de São Paulo-Escola Paulista de Medicina.

¹ Professora Adjunta do Departamento de Cirurgia - Disciplina de Técnica Operatória e Cirurgia Experimental - UNIFESP - EPM.

² Aluno da Graduação em Medicina vinculado ao Programa de Iniciação Científica da Disciplina de Técnica Operatória e Cirurgia Experimental-UNIFESP-EPM.

³ Professora Doutora Adjunta da Disciplina de Parasitologia-UNIFESP-EPM.

⁴ Professor Adjunto da Disciplina de Técnica Operatória e Cirurgia Experimental-UNIFESP-EPM.

⁵ Professor Titular do Departamento de Cirurgia - Disciplina de Técnica Operatória e Cirurgia Experimental-UNIFESP-EPM.

Publication Dates

• Publication in this collection
  14 Mar 2001
• Date of issue
  2000