Effect of glucocorticoids on surface IL-10 receptors and intracellular IL-10 in mouse splenocytes, thymocytes, monocytes, and dendritic cells: immunofluorescence, confocal microscopy, and electrophoresis study

ABSTRACT

Glucocorticoids are capable of stimulating the secretion of interleukin -10 [IL-10] by leukocytes. The probable role of glucocorticoids in the susceptibility of immune cells to IL-10 mediated actions has not yet been studied. In this study, we examine the expression of IL-10 and IL-10 receptors [IL-1OR] in mouse splenocytes, thymocytes, monocytes, and dendritic cells. In addition, we determined the effects of the glucocorticoid [methylprednisolone] on IL-10 secretion and IL-10R expression in the previous immune cells. Forty-eight male mice with the C57BL/6 genetic background were used. The animals were 10-12 weeks of age and were divided equally into two groups. The animals in group I were provided with a balanced standard diet and water ad libitum. The animals in group II were provided with a balanced standard diet and methylprednisolone [o.5mg/kg/day] in the drinking water daily for 7 days. The mice in groups I and II were sacrificed after 7 days and the spleen and thymun were removed to isolate the splenocytes and thymocytes. The blood was also aspirated to isolate monocytes. Group III included mice with the mouse dendritic cell line [JAWS II]. Cells of the mice in this group were divided into two subgroups. Group IIIa included the dendritic cells cultured in Iscove's modified Dulbecco's medium and group IIIb included the dendritic cells cultured in Iscove's modified Dulbecco's medium supplemented with methylprednisolone [2micro g/ml] for 6h. All the previous cells were processed for immunohistochemistry, western blotting, immunoassay analysis, and electrophoresis. All the cells examined showed an intense fluorescent surface expression of IL-10R as detected by the immunofluorescence technique. On using methylprednisolone, there were few and faint expressions of IL-1OR. The intracellular IL-10 molecules were few in the dendritic cells but after treatment with methylprednisolone, the IL-10 molecules were numerous and intense. Western blotting and electrophoresis techniques showed intense precipitation of IL-10 at 35 kDa and faint precipitation of IL-10R at 110kDa in all the cells examined after glucocorticoid treatment. There was also a highly significant [P<0.001] increase in the level of IL-10 in all the cells examined after using methylprednisolone as detected by immunoassay. Our findings could contribute toward understanding the immunomodulating mechanism of the action of glucocorticoids, which may result in greater therapeutic benefit in some diseases by enhancing the synthesis of the anti-inflammatory cytokine IL-10 and decreasing the expression of IL-1OR in some immune cells