Extracellular presence of IL-8 in the astrocyte-rich cultured cerebellar granule cells under acidosis

Abstract

In order to evaluate the functional role of chemotactic cytokines in the regulation of brain function, we examined the effects of acidosis on the production of IL-8 in cultured neurons and/or astrocyte-rich cerebellar granule cells as assessed by the ELISA method. A time-dependent and significant production of IL-8 was detected in the extracellular fluid of astrocyte-rich cultured cells at 2, 3 and 6 hrs after treatment with acidified Krebs-HEPES buffer (pH 6.9), although such production did not appear in the fluid of neuron-rich cells. Additionally, microglia were detected by microscopic examination in both cultured cells under acidotic conditions. Only astrocyte-containing cultured cells produced a marked increase in intracellular IL-8 under acidotic conditions, although this production was much less than that seen in the extracellular fluid at 6 hrs under acidosis. The increase of IL-8 in astrocyte-rich cultures induced by acidosis was potentiated by treatment with glutamate, which enhanced the increase of cytosolic Ca 2+ levels under acidosis, and was affected by extracellular Ca 2+ conditions, by cyclosporine A, an inhibitor of calcineurin, and by trifluoperazine, an inhibitor of phospholipase A 2. Significant inhibition of IL-8 production was detected after 6 hrs of pretreatment with trifluoperazine. Furthermore, the production of IL-8 under acidosis was associated with the appearance of astrocyte damage. These results suggest that Ca 2+-dependent IL-8 is produced by astrocytes, but not neuronal cells, under acidosis, and that this production may be related to the process of cell dysfunction resulting from membrane destruction induced by acidosis.