| Glucocorticoids (GCs) regulate a variety of growth, metabolic, developmental, and immune functions and play a pivotal role in preserving basal and stress-related homeostasis. They also represent one of the most widely prescribed anti-inflammatory and immunomodulatory drugs during the last 60 years. The molecular mechanisms of GCs have been extensively studied. However, the molecular mechanism is still largely unclear. It is believed traditionally that GCs exert most of their effects genomically. Upon ligand binding, glucocorticoids receptor (GCR) translocates into the nucleus of cells, where it modulates the transcriptional activity of glucocorticoid-responsive genes in either of two ways: by binding to specific sequences in the promoter region of target genes, the glucocorticoid-response elements (GREs), or through protein-protein interactions with other transcription factors, such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and several signal transducers and activators of transcription (STATs). Latest findings suggest that genomic activity might become relatively more important in mediating the therapeutic effects of high-dose pulsed glucocorticoid activity. Non-genomic effects are charactared as short latency, and insensitive to inhibitors of DNA transcription or protein synthesis (e.g. actinomycin D or cycloheximide) compared to genomic effects. Though GCs are widely used in the treatment of inflammatory and autoimmune states, studies of genomic effects are mainly performed in the fields of neuroendocrinology. Latest findings suggest that genomic activity might become relatively more important in mediating the therapeutic effects of high-dose pulsed glucocorticoid activity. However, there are few genomic anti-inflammatory effects reported.Human polymorphonuclear neutrophils (PMN) comprise 50-70% of circulating leukocytes. In the inflammatory response, few cells play as prominent a role as the neutrophil. Human neutrophil contains three main lysosomal granules, azurophil granules, specific granules and gelatinase granules, and secretory vesicles. Specificproteolytic and digestive enzymes capable of destroying extracellular matrix and bacterial debris are stored inside these granules, which therefore are involved in immune and inflammatory processes as well as in a variety of diseases and tissue injuries, such as rheumatoid arthritis, acute spinal cord trauma, acute lung injury and chronic obstructive pulmonary disease (COPD). These diseases are indications of glucocorticoid pulse therapy.In the present study, human neutrophils were isolated by the discontinuous saline-Percoll gradient technique. Purified human neutrophils were pretreated with GCs for 5 minutes, and then primed with N-formyl-methionyl-leucyl- phenylalanine (fMLP) (10"6 M) or phorbol myristate acetate (PMA) (50 ng/ml) in the presence of cytochalasin B (CB). The release of markers of neutrophil granules, myeloperoxidase (MPO) lactoferrin (LF) and lysozyme (LZ), was measured by enzymology and ELISA methods respectively. Morphological changes of PMA stimulated PMN degranulation affected by methylprednisolone were observed and photographed under inverted phase contrast microscope. Intracellular levels of cyclic AMP (cAMP) were measured by a radioimmunoassay, and cytosolic free Ca2+ ([Ca2+]i) was measured by using a fluorescent microscopic imaging technique. The main results as follows:1. In the presence of CB, GCs showed significant inhibitory effects on MPO, LF and LZ release during neutrophil degranulation in 5 minutes after fMLP or PMA administration. It required higher concentrations of hydrocortisone than Methylprednisolone to inhibit the degranulation. Neither glucocorticoid receptor antagonist RU486 nor the protein synthesis inhibitor cycloheximide could alter the inhibitory effects of GCs. RU486, cycloheximide or ethanol alone has not this inhibitory effect.2. Morphological changes of PMA stimulated PMN degranulation were blocked by Methylprednisolone. Methylprednisolone inhibited pseudopod extension and plasmatorrhexis stimulated by PMA, thus maintained the normal shape of PMN.3. GCs can elevate intracellular levels of cAMP. Neither glucocorticoid receptor antagonist RU486 nor the protein synthesis inhibitor cycloheximide could alter... |
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