Cytoprotective effect of low-dose tacrolimus on islets of Langerhans in cultures subjected to stimulation by acute rejection cytokines

Balibrea del Castillo, José María; Arias-Díaz, Javier; García Martín, María Cruz; Vives-Pi, Marta; García Pérez, Juan Carlos; Cid, Ramón Cantero; Ameigeiras, Elena Vara; Balibrea Cantero, José Luis

doi:10.1016/S2173-5077(10)70184-8

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Abstract

Introduction

The improvement in pancreatic islet transplantation results is due to immunosuppression protocols that include, among others, low-dose tacrolimus. Both antiinflammatory and anti-oxidant effects of tacrolimus could be useful in preventing primary rejection.

Aim

To evaluate in vitro islet low-dose tacrolimus response after pro-inflammatory stimulation.

Material and methods

Isolated rat islets were cultured in RPMI medium in the presence of IL-1 (50 UI/ml) plus IF-γ(1000UI/ml) and tacrolimus (5 ng/ml). The 24h production of lipoperoxide (LPO) and nitric oxide (NO) were measured as oxidative stress markers. Determination of apoptosis markers (nucleosome content and Bcl-2) was also performed.

Results

Oxidative stress (LPO 10.1±1.16 pmol/isletx24; NO 19.1±3.28 pmol/isletx24h) and apoptosis (nucleosome 0.24±0.04 UI/islet; Bcl-2 0.69±0.212 UI/islet) markers showed a very significant increase after cytokine stimulation (P<.01). Both effects improved by adding tacrolimus to the medium. Protective effect was complete when lipoperoxide (1.58 pmol/isletx24 h), nitric oxide (9.81 pmol/isletx24 h) and Bcl-2 (1.37±0.23 UI/islet) were determined.

Conclusion

In vitro cytoprotective effect of low-dose tacrolimus on isolated rat islets decreases both oxidative stress and apoptosis markers after stimulation of proinflammatory mediators.

Keywords:

Pancreatic islet transplantation

Apoptosis

Oxidative stress

Tacrolimus

Immunosuppression

Resumen

Introducción

La mejoría de los resultados en el trasplante de islotes pancreáticos se debe en gran parte a la introducción de nuevos protocolos de inmunosupresión que incluyen, entre otros, tacrolimus a bajas dosis. Este fármaco tiene efectos antioxidantes y antiapoptóticos que podrían ser de utilidad en la prevención del rechazo primario.

Objetivos

Evaluar la respuesta in vitro a tacrolimus a bajas dosis en islotes de rata estimulados con citocinas proinflamatorias implicadas en el rechazo primario de islotes.

Material y método

Se cultivaron islotes de rata en medio RPMI determinándose producción de lipoperóxido (LPO) y óxido nítrico (NO) y marcadores de apoptosis (nucleosomas y Bcl-2) en presencia de IL-1 (50UI/ml) e IF-γ(1000UI/ml) y adición de tacrolimus (FK-506; 5ng/ml).

Resultados

Tras la estimulación se apreció un aumento muy significativo (p < 0,01) de los marcadores de estrés oxidativo (LPO 10,1±1,16 pmol/islote x 24; NO 19,1±3,28 pmol/islote x 24 h) y apoptosis (nucleosomas 0,24±0,04; Bcl-2 0,69±0,212). Dichos efectos fueron contrarrestados de manera significativa tras añadir tacrolimus, siendo la reversión completa (p NS frente a controles) en el caso de la producción de lipoperóxidos (1,58 pmol/islote x 24h) y óxido nítrico (9,81 pmol/islote x 24h), así como en el descenso de Bcl-2 (1,37±0,23 UI/islote).

Conclusiones

El efecto citoprotector in vitro del tacrolimus a bajas dosis sobre islotes estimulados con citocinas proinflamatorias consigue aminorar la generación de estrés oxidativo y la activación de la apoptosis, habitualmente implicados en el rechazo en las primeras 48 h postimplante.

Palabras clave:

Trasplante de islotes pancreáticos

Apoptosis

Estrés oxidativo

Tacrolimus

Inmunosupresión

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