Rapid Inhibitory Effects Of Glucocorticoids On Human Neutrophil Degranulation And Its Molecular Mechanism

Glucocorticoids (GCs) are secreted by the adrenal cortex and regulate a variety of growth, metabolic, developmental, and immune functions and play a pivotal role in preserving basal and stress-related homeostasis. They also used widely as anti-inflammatory and immunomodulatory drugs at clinical treament. Glucocorticoids can exert their effects on genomic and non-genomic action . The mechanism of the genomic actions of glucocorticoids has been fairly well investigated. As lipophilic substances, glucocorticoids pass very easily through the cell membrane into the cell, where they bind to the ubiquitously expressed cytosolic glucocorticoid receptor. The activated glucocorticoid–receptor complex is then translocated into the nucleus where it binds to specific DNA sites. This initiates or inhibits transcription of certain genes and hence the synthesis of various proteins. It is important to know that these genomic effects occur at any relevant therapeutic concentration and are usually observed not earlier then 30 min after the receptor binding.There is no doubt that the therapeutic effects of glucocorticoids are mostly receptor-mediated. However, there is growing evidence that there are also rapid non-genomic glucocorticoid effects. Non-genomic effects are charactared as short latency, and insensitive to inhibitors of DNA transcription or protein synthesis compared to genomic effects.Much less is known about the molecular mechanism of non-genomic effects,although their existence has been apparent from the very beginning of research into the physiology and pharmacology of steroids.Human polymorphonuclear neutrophils (PMN) are very important in the host defense,in response to diverse stimuli,neutrophils are activated and secrete a series of lytotoxins,such as o2- and granual protease,to destroy extracellular matrix and bacterial debris.but when activated extensively,neutrophils can lead to a variety of diseases and tissue injuries,such as rheumatoid arthritis,ischemia-reper fusion injury,chronic obstructive pulmonary diseases and asthma.suppressing the extensive or inappropriate activation of neutrophils can ameliorate these inflammatory diseses. Human neutrophil contains three main lysosomal granules, azurophil granules, specific granules and gelatinase granules, and secretory vesicles. Specific proteolytic 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. In our previously study, we found that high-dose glucocorticoids can inhibit the release of these three lysosomal granules of human neutrophil in 5 min, RU486 and CHX can't reverse its effest ,it was a non-genomic action,but the signal transduction pathways are still unclear.The mechanisms of neutrophil degranulation are very complicated ,it is known that several signal transduction pathways involved in it and cAMP-PKA is a very important pathway. cAMP is a very important second messenger in regulating neutrophil functions, it is formed from ATP by the action of the enzyme, adenylyl cyclase (AC), and is degraded by a family of cAMP-specific phosphodiesterase (PDE)enzymes, which catalyzes the hydrolysis of cAMP to inactive 5'-AMP. Elevation of intracellular cAMP levels is believed to suppress the activation of neutrophils. For example, adenosine,which activates the Gas protein to stimulate AC via occupancy of A2a receptors on neutrophils, has been widely recognized to diminish the inflammatory response. in most of inflammatory cells, including human neutrophils,the PDE belongs to the PDE4 family.so either activating the activity of AC or inhibiting the activity of PDE can elevate the intracellular cAMP level,then suppress neutrophils degranulation.In the present study, human neutrophil isolation was performed using a modification of discontinuous saline-Percoll gradient technique.Purified cells were pretreated with PKA inhibitor H89,stimulating G protien inhibitor GDPβs or MβCD respectively before added with GCs,and then stimulated with fMLP.The markers of neutrophils granules, myeloperoxidase (MPO) and lactoferrin(LF),was measured by enzymology and ELISA methods.Intracellular levels of cAMP were measured by radioimmunoassay, PDE activity was assayed using a commercially available assay kit.The main results as follows:1. Glucocorticoids induced a substantial increase in cAMP levels of PMN.2. The inhibitory effect of glucocorticoids was reversed by protein kinase A inhibitor H89.3.The inhibitor of stimulating G protein GDPβs can't reverse the inhibitory effect of glucocorticoids on permeable neutrophil degranulation.4. fMLP enhance the activity of PDE of neutrophils,and GCs inhibit the PDE activity.5. MβCD can't reverse the inhibitory effect of glucocorticoids on human neutrophil degranulation.In summary, these results indicate that the inhibitory effect of glucocorticoids on neutrophils degranulation is cAMP-PKA dependent, it occurs through inhibiting the PDE activity but not through stimulating the AC activity of neutrophils,and the lipid raft might be not involved in it.We found one signal transduction pathway of GCs inhibit neutrophils degranulation.