Methyl-prednisolone up-regulates monocyte interleukin-10 production in stimulated whole blood

Glucocorticosteroids (GCS) have been used successfully in the treatment of inflammatory conditions such as asthma and acute graft-vs-host disease, but their mode of action remains unclear. There have been numerous reports of the in-vitro suppression of cytokine production by GCS based on quantitatio n of cytokines by ELISA on bull; supernatants from isolated cell culture sy stems. We report the use of a whole-blood intracellular cytokine assay whic h is more representative of an in-vivo environment. We examined the effects of GCS, prednisolone and dexamethasone, on cytokine production by individu al cells (monocytes, T lymphocytes and natural killer or NK cells) in heter ogenous cell populations. Cells in whole blood were activated with various stimuli: phorbol ester and calcium ionophore for T cells, Escherichia coli lipopolysaccharide (LPS) for monocytes, and phytohaemagglutinin (PHA) plus interleukin (IL)-12 for NK cells. Brefeldin A was used as an intracellular transport inhibitor to enhance the detection of intracellular cytokine prod uction. The effects of various concentrations (10(-5), 10(-7), 10(-9) and 1 0(-11) M) of GCS on cytokine production were studied using multiparameter f low cytometry. After surface staining with fluorescently conjugated monoclo nal antibodies (MoAbs) to identify cell type, cells were fixed and permeabi lised. Intracellular cytokines interferon (IFN)-gamma, IL-10, IL-1 alpha an d beta, IL-2, tumour necrosis factor (TNF)-alpha, and IL-12 were stained wi th their respective conjugated MoAbs. The GCS both caused a dose-dependent modulation of cytokine production by T cells, monocytes and NK cells. After 4 h, a decrease in the MFI (amount of cytokine produced per cell) was note d for all cell types. After 24 h a decrease in both MFI and the percentage of cells producing cytokine was observed for all cell types. The exception was monocyte production of IL-10 which was enhanced at low concentrations o f GCS (10(-9) and 10(-11) M). Our findings thus suggest that one anti-infla mmatory mechanism of GCS action may be through inhibition of the release of pro-inflammatory cytokines IL-1 alpha and beta, IL-2, IFN-gamma and TNF-al pha, and up-regulation of the anti-inflammatory cytokine IL-10.