1.Regulation Of H₂S Production By Glucocorticoids In Macrophages 2.Endotoxin Tolerance Of Adrenal Gland: Attenuation Of Corticosterone Production In Response To Lipopolysaccharide And Adrenocorticotropin Hormone

In severe infectious diseases, inflammatory responses strongly activate the HPA axis and stimulate the release of adrenocorticotropic hormone (ACTH), which in turn stimulates the secretion of glucocorticoids (GCs) from the adrenal cortex. It is well known that GCs have anti-inflammatory effects, which is the main reason for the clinical usage of GCs to treat acute and chronic inflammatory diseases. However, GCs have also been shown to exert pro-inflammatory effects in some cases. Scientists have proposed that GCs could balance between pro- and anti-inflammatory mediators and control their own strength, so as to reduce the side effects of GCs. So far, the mechanisms involved remain to be further elucidated.Although GCs play an important role in maintaining the homeostasis during inflammation, reversible adrenal insufficiency has been frequently diagnosed in critically ill patients with sepsis who have either low basal cortisol levels or low cortisol responses to adrenocorticotrophic hormone (ACTH) stimulation. Moreover, corticosteroid insufficiency is always associated with a high mortality rate. Therefore, it seems to be of great significance to clarify the mechanisms responsible for the occurrence of adrenal insufficiency in severe inflammatory diseases.In the first part of present study, we found that macrophages produced not only the well-known pro-inflammatory gaseous transmitter nitric oxide (NO), but also the newly recognized gas molecule hydrogen sulfide (H2S). In addition, H2S exerts certain anti-inflammatory effects by inhibiting NO production. We also found that GCs decreased the production of both NO and H2S, and we further investigated the mechanisms involved in the inhibitory effects of GCs on H2S production. These findings provide important experimental evidence for the mechanisms by which GCs coordinate the balance between pro- and anti-inflammatory mediators during inflammation.It was found that prior exposure of innate immune cells like monocytes/ macrophages to minute amounts of endotoxin caused them to become refractory to subsequent endotoxin challenge, a phenomenon called "endotoxin tolerance". Now, it is generally accepted that endotoxin tolerance contributes to immunosuppression during sepsis. Therefore, in the second part of this study, we hypothesized that endotoxin tolerance might have occurred in the adrenal gland and led to hyporesponsiveness of adrenal gland during sepsis. To test this, we firstly observed the effect of LPS on corticosterone production in the rat adrenal zona fasciculata-reticularis (F/R) cells and explored the synthetic pathways involved. Then, we studied the effect of LPS pretreatment on LPS- and ACTH-induced corticosterone production in F/R cells.Main results:1.Capacity of H2S biosynthesis in macrophages and the local functions of endogenous H2S1)Macrophages expressed H2S-forming enzyme cystathionine-gamma-lyase (CSE) and produced H2S.2)Lipopolysaccharide (LPS) increased CSE expression and H2S production rate. This suggests that macrophages be one of H2S producing sources during Gram-negative bacteria-induced inflammation.3)L-cysteine reduced LPS-induced nitric oxide (NO) production. CSE inhibitor blocked the inhibitory effect of L-cysteine. CSE knockdown increased, whereas CSE overexpression decreased LPS-induced NO production. Thus, it would suggest that H2S might exert anti-inflammatory effects by inhibiting NO production in macrophages during LPS-induced inflammation.2.Regulation of H2S Production by Glucocorticoids in Macrophages1)Dexamethasone suppressed LPS-induced CSE expression and H2S production rate as well NO production. This may be a mechanism by which glucocorticoids coordinate the balance between pro- and anti-inflammatory mediators during inflammation.2)L-arginine increased whereas NG-nitro-L-arginine methyl ester (L-NAME) decreased LPS-induced CSE expression and H2S production. These suggest that NO is an important endogenous stimulus in the formation of H2S during LPS-induced inflammation.3)Dexamethasone plus L-NAME significantly decreased LPS-induced CSE expression and H2S production compared to L-NAME. This suggests that dexamethasone may directly inhibit CSE expression and H2S production besides NO-dependent way.4)Dexamethasone reduced CSE mRNA stability in primary macrophages, and our previous resultes sugguested that dexamethasone decreased CSE promoter activity in RAW 264.7 macrophages. These suggest that dexamethasone inhibition of CSE expression is through different mechanisms in primary macrophages and macrophage cell line and the mechanism of action of glucocorticoid on CSE expression might be related to cell types. 3. Regulation of corticosterone production by LPS in rat primary F/R cells.1)Toll-like receptor 4 (TLR4) and P450 11β-hydroxylase (CYP11B1) were co-localized in adrenal gland and primary F/R cells.2)LPS stimulates corticosterone production and expression of StAR and CYP11B1 in primary F/R cells, but did not affect the mRNA and protein levels of CYP11A1. ACTH dose-dependently increased both mRNA and protein levels of StAR and all the synthetic enzymes, CYP11A1, HSD3B, CYP21 and CYP11B1. Notably, the synthetic enzymes involved in ACTH stimulation differed from those in LPS stimulation, which indicates that the intracellular signaling mechanisms responsible for ACTH and LPS stimulation of corticosteroid production may be different.4. Regulation of LPS/ACTH-induced corticosterone production by LPS pretreatment.1)LPS pretreatment caused a significant decrease in corticosterone production in response to subsequent LPS or ACTH stimulation in primary F/R cells. These results indicated that endotoxin tolerance might also occur in adrenal gland during sepsis, and endotoxin tolerance of adrenal gland during sepsis is one of mechanisms for adrenocortical insufficiency.2) We found that TLR4 expression was significantly decreased by LPS over a 24 h period of treatment, which was consistent with down-regulation of corticosterone production and the expression of StAR and CYP11B1 in response to second stimulation of LPS. These data indicate that reduced TLR4 expression may account for hypo-responsiveness to second stimulation of LPS in adrenal gland.3)The present study also demonstrated that LPS caused decreased ACTH-R in F/R cells, and LPS pretreatment decreased expression of some(CYP11A1,HSD3B,CYP21), but not all steroidogenic enzymes and proteins, suggesting that reduced ACTH-R in adrenal gland just partly account for hypo-responsiveness to ACTH, and additional mechanisms whereby LPS pretreatment induces hypo-responsiveness to ACTH in adrenal gland might exist.4) The present study indicates that LPS pretreatment suppress both LPS- and ACTH-induced expression of steroidogenic enzymes. The stored GCs can maintain normal rates of secretion for only a few minutes in the absence of continuing biosynthesis. Therefore, any disruption in glucocorticoid biosynthesis will immediately affect the response of adrenal cortex to various stresses. Our findings suggest that induction and activation of these steroid metabolic enzymes can be a promising treatment strategy for adrenocortical insufficiency during sepsis.Conclusions:Our results suggest that macrophages are one of H2S producing sources. H2S might exert anti-inflammatory effects by inhibiting NO production. Dexamethasone may directly inhibit CSE expression and H2S production besides NO-dependent way. Inhibition of H2S and NO production may be a mechanism by which glucocorticoids coordinate the balance between pro- and anti-inflammatory mediators during inflammation.Endotoxin tolerance of adrenal gland during sepsis is one of mechanisms for adrenocortical insufficiency, and activation of synthetic enzymes can be a promising treatment strategy.