The Regulation Of Interleukin-8 Secretion Induced By The Aldosterone To Human Mononuclear Macrophage THP-1

Background and Objective:Aldosterone, a major member of the rennin-angiotensin-aldosterone system (RAAS), which is recognized as a major regulator of ionic homeostasis by modulating cation transport, mainly exists in kidney and colon. Meanwhile, several studies showed that the RAAS was activated when the body was seriously stressed and there were increased aldosterone levels found in these patients. For a long time, it was assumed that aldosterone is a promoter of hypertension and myocardial fibrosis, can promote the proliferation of cardiac fibroblast and collagen synthesis, resulting in myocardial remodelling and cardiac dysfunction. It was assumed that aldosterone can act via the classic genomic mechanism involving binding to the intracellular receptor, the mineralocorticoid receptor (MR), thereby affecting transcription and protein synthesis. Yet, many expriments have shown that aldosterone also elicits nongenomic responses by affecting signal transduction[1]. Toll-like receptors (TLRs) are an evolutionarily conserved family of pattern recognition receptors central to the innate immune response to infection, and have an important role in innate immune responses and subsequent activation of acquired immune responses. TLRs are responsive to pathogen-associated ligands such as lipopolysaccharide (LPS) and other microbial components. Recent evidence also suggests that TLRs can recognize endogenous ligands that signal host injury, including hyaluronic acid, heparan sulfate, heat-shock proteins, fibronectin, and biglycan'21. In this study we test the idea that TLR4 recognize the aldosterone as an endogenous ligand or TLR4 is a novel type of aldosterone receptors.Method:We mimicked the stress situation by add the aldosterone to the cells. Before stimulation experiments, THP-1 cells were grown to 90% confluence in 75 cm2 plastic culture dishes and were serum starved for 2h. Aldosterone prediluted to 104nM,103nM,102nM and 101nM final concentration was directly added to medium without 10% fetal calf serum, and after gentle swirling, the cells were incubated for 5min,30min,2h,6h and 12h unless otherwise indicated. Controls were stimulated with medium without 10% fetal calf serum. In case of treatment with inhibitors, appropriate predilutions were directly added to cells prior to the addition of aldosterone unless other indicated. The preincubation times for the different inbibitors used are indicated as 30 min. At indicated time-points after the treatment, cells were harvested and washed twice with cold PBS; cell proteins were extracted using cell lysis buffter and the expression of MR and TLR4 were measured by western bloting. Realtime-PCR was used to determine mRNA levels of IL-8, MR and TLR4. IL-8 released in the conditional medium was used as a means of evaluating inflammation and was quantified with enzyme-linked immunosorbent assay (ELISA) kits according to the manufacture's instruction. Data are presented as mean±SEM, unless otherwise stated. Differences between treatment groups were determined by Student's t test. P value less than 0.05 is considered statistically significant.Result:By using a specific antibody which recognizes the IL-8, we analyzed the levels of IL-8 in THP-1 cultures after treatment with aldosterone by ELISA. Follwing ALD stimulation, the levels of IL-8 were increased until 2 h and further increased as time passed, implicating a possibility that ALD may induce IL-8 production in a time-dependent manner, and may act via the classic genomic mechanism, but there is no synergetic effect to IL-8 production induced by LPS. Spironolactone, the classic MR antagonists, significantly reduced ALD-induced cytokine IL-8 production in cultured THP-1 cells. Similarly, compared with ALD treated cells, anti-TLR4 antibody-treated cells had lower IL-8 levels in the conditional medium and in mRNA expression. Western blot analysis showed upregulation of TLR4 protein expression after 102 nM ALD treatment, indicated ALD may involve the immunoregulation via TLR4 recognization. Collectively, these these data demonstrate that ALD may mediate the crosstalk of immune system and RAAS in responses to stress and injury and a critical role for an ALD-TLR4 pathway in this pathway. Conclution:ALD induced IL-8 production in a time-dependent manner, and may act via the classic genomic mechanism. The treatment of spironolactone and anti-TLR4 antibody blocked the upregulation of IL-8 induced by aldosterone. Aldosterone increased TLR4 protein expression after the treatment of 12h. So aldosterone plays an important role on inflammation reaction induced by stress. The mechanism may be involved the activating of neuroendocrine and innate immune response. Importantly, we think that TLR4 may recognize the aldosterone as an endogenous ligand or TLR4 maybe a novel type of aldosterone receptors.