Brain Mast Cells Regulate Microglial Activation

Background:Microglia are the resident immune cells in the central nervous system(CNS),about 10-20%of the total number of glial cells in the brain.Microglia are involved in monitoring and regulating neuronal survival in the brain,and play a pivotal role in the CNS damage,repair,and development.When subject to abnormal stimulations,such as neurotoxins,neuronal debris,or injury,microglia gradually become activated and produce inflammatory mediators,including tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),interleukin-6(IL-6),prostaglandins E2(PGE2),nitric oxide(NO)and ROS.Accumulation of these proinflammatory and cytotoxic mediators is directly deleterious to the neurons and subsequently induces further activation of microglia,resulting in a vicious cycle.Thus,inhibition of the microglial activation and the subsequent inflammatory process may help identify novel therapeutic strategies to eliminate the deleterious effects of microglia.Besides releasing proinflammatory mediators,microglia also respond to proinflammatory signals released from other non-neuronal cells of immune origin.In this context,mast cells are particular relevance.Mast cells are multifunctional effector cells of the innate immune system.They are infamous and well studied for their role in immunoglobulin E(IgE)-associated allergic and inflammatory disorders,but have not been examined much in the brain.In the mammalian brain,mast cells are typically found in the thalamus,hypothalamus,hippocampus,meninges and choroid plexus.Mast cells generally reside on the brain side of blood-brain barrier(BBB).Under normal circumstances,mast cells around the BBB with a small portion degranulating,including histamine,tryptase,and other mediators to maintain the normal function of the nervous system,such as regulating the secretion of hormones,mood,feeling and cognition.Meanwhile,the brain mast cells are also involved in the pathological changes of the CNS disease,such as autism,Parkinson's disease,Alzheimer's disease,multiple sclerosis and cerebral ischemia.They lie in close proximity to the basal side of the blood vessel wall,thus,can capture the immune signals in the first time,even before microglia.Mast cell granules bear numerous preformed and newly synthesized reactive chemicals,termed 'MC mediators',many of which are active in the CNS or peripheral nervous system.Rapid release of potent preformed mediators is quickly followed by synthesis of lipid mediators and a slower,de novo synthesis of cytokines and chemokines.Thus,mast cells are both sensors and effectors in communication among nervous,vascular and immune system.However,the direct effect of mast cells on microglial activation has not been reported.Objective:To investigate the protein expression of all of the four histamine receptors on microglia and the mechanism of histamine-induced microglial activation.Method:1.Western blot,immunofluorescence and flow cytometry were used to detect the protein expression of four histamine receptors on microglia.2.Firstly,microglia was exposed to various concentration of histamine(0.001,0.01,0.1 and 1 ?g/ml)for 24 hours.Flow cytometry was used to detect the expression of four histamine receptors and ED8 on microglia.Immunofluorescence was also used to detect the expression of ED8.ELISA was used to detect the production of TNF-? and IL-6 in culture medium.Secondly,microglia were exposed to 0.1 ?g/ml histamine for 0.5,2,6 and 24 h.Flow cytometry was used to detect the expression of four histamine receptors;ELISA was used to detect the production of TNF-? and IL-6 in culture medium.Then,before exposed to 0.1 ?g/ml histamine for 24 h,microglia was preincubated with 10 ?M antagonist of four histamine receptors.ELISA was used to detect the production of TNF-? and IL-6 in culture medium;flow cytometry was used to detect the mitochondrial membrane potential in microglia.3.Microglia was exposed to various concentration of agonists of four histamine receptors(0.1,1,10 and 100?M)for 24 h.ELISA was used to detect the production of TNF-? and IL-6 in culture medium4.Cultured microglia was treated with 0.1 ?g/ml histamine for 15,30,60,120 and 240 min,respectively.Or,microglia was pretreated with 10?M antagonist of H₁R and H₄R for 30 min and then exposed to histamine(0.1 ?g/ml)for 15 or 60 min.Western blot was used to detect the phosphorylation of AKT and MAPK signaling pathway.Based on the results of the previous step,Microglia was pretreated with the inhibitors of signaling pathways SP600125(10 ?M),SB203580(10?M)and Wortmannin(1 ?M)for 30 min before being challenged with histamine(0.1 ?g/ml)for 24 h;the cell suspensions were collected for ELISA.5.Microglia was pretreated with the inhibitor of signaling pathways Wortmannin(1 ?M),antagonists of H₁R and H₄R,and PDTC for 30 min before being challenged with histamine(0.1 ?g/ml)for 24 h;the cell suspensions were collected for ELISA.Immunofluorescence was used to detect the expression of NF-?B(p65);Western blot was used to detect the phosphorylation of I?B?.Results:1.Western blot,immunofluorescence and flow cytometry analysis confirmed that primary rat microglial cells express histamine H1,H2,H3 and H4 receptors.2.Histamine could promote an increase in the expression of H₁R and H₄R on microglial cells in a dose-dependent and time-dependent manner;histamine in a dose-dependent manner increased the expression of ED8 on microglial cells;histamine in a dose-dependent and time-dependent manner increased the production of TNF-? and IL-6 from microglia;antagonists of H₁R and H₄R receptors could inhibit TNF-? and IL-6 production of histamine-stimulated microglia;histamine could induce microglia mitochondrial membrane potential loss,and antagonists of H₁R and H₄R receptors could inhibit histamine-induced mitochondrial membrane potential loss.3.Agonists of H₁R and H₄R receptors could stimulate microglia produces TNF-?and IL-6,and in a dose-dependent manner,whereas pretreatment with the antagonists of H₁R and H₄R receptors could block the production of TNF-? and IL-6.4.Histamine could activate AKT,p38 MAPK and JNK MAPK signaling pathway;however,pretreatment with the antagonists of H₁R and H₄R receptors could inhibit the effects.Pretreatment with AKT,p38 and JNK inhibitors could partially block histamine-induced production of TNF-? and IL-65.Histamine could induce microglial NF-?B(p65)into the nucleus,and antagonists of AKT,H₁R and H₄R could block this effect;histamine-induced phosphorylation of I?B? could be inhibited by antagonists of AKT,H₁R and H₄R.NF-?B inhibitor PDTC could inhibit histamine-induced production of TNF-? and IL-6.Conclusion:(1)Microglia express histamine H1,H2,H3 and H4 receptors.(2)Histamine upregulates expression of H₁R and H₄R.(3)Agonists of H₁R and H₄R coud stimulate the prodyction of TNF-? and IL-6 from microglia.(4)Histamine could induce microglial activation and TNF-? and IL-6 production.(5)Histamine induced inflammatory factors releasing is related to mitochondrial membrane potential.(6)Histamine induces activation of microglia via H₁R and H₄R-MAPK and PI3K/AKT-NF-?B pathway.Section I The effects of brain mast cell degranulation on microglial activation and CNS inflammation in vivo.Objective:To investigate the brain mast cell degranulation on microglial activation and CNS inflammation in vivo.Method:Mast cell stabilizer cromolyn(Cro)and/or degranulator compound 48/80(C48/80)were injected into the rat right hypothalamus.1.SD rats were divided into six groups:Control group,C48/80 group,C48/80+Cro100?g group,C48/80+Cro200?g group,Cro100?g group and Cro200?g group.Animals received 2.5 of 1?g/?l C48/80(2.5 ?g)or 2.5 ?l 0.9%NaCl directly in the hypothalamus.Animals remained in their cages for 30 min or 24 h and were not restrained.To stabilize mast cells,the same site was pretreated with 1 ?l of cromolyn(100 or 200 ?g/?l)30 min before C48/80 administration.Control animals were pretreated with 1 ?l 0.9%NaCl.Rats were killed after drug administration,and their brains,followed by the separation of meninges from which,were used for morphological(n=6)and biochemical(n=6)analyses.2.The mice were divided into four treatment groups.Of the wild-type(WT)mice groups,one group received C48/80 administration(n=5)and a control group received saline(n=5).Similarly with the KitW-sh/W-sh mice,one group received C48/80 administration(n=5)while the other group received saline(n=5).Results:1.Cromolyn repressed C48/80-induced mast cell activation in hypothalamus.Stabilization of mast cell inhibited microglial activation in hypothalamus.Stabilization of mast cell inhibited C48/80-induced TNF-? and IL-6 production.Stabilization of mast cell depressed MAPK and PI3K/AKT activation in hypothalamus.Cromolyn inhibited activated mast cells which induced receptor change on microglia in hypothalamus2.C48/80 had no effect on microglial activation in hypothalamus of KitW-sh/W-sh miceSection ? The effects of brain mast cell degranulation on microglial activation and CNS inflammation in vitro.Objective:To investigate the effects of HMC-1 degranulation on microglial activation.Methods:HMC-1 and primary microglial cells were used for co-culture.1.HMC-1 was exposed to various concentration of CRH(1,101,102,103,104 nM)for 12,24,48 and 72 h,respectively.Flow cytometry was used to detect Fc receptor of mast cell.Then,HMC-1 was exposed to 100 nM CRH for 12,24,48 and 72 h,the expression of TNF-? and IL-6 in cell supernatants were detected by ELISA.2.HMC-1 was stimulated with PBS or 100 nM CRH for 12,24,48 and 72 h,then the culture medium of HMC-1 were taken for incubating microglia for 12 or 24 h,flow cytometry and immunofluorescence were used to detect microglial activation.3.Firstly,microglia was exposed to 100 nM CRH for 12,24,48 and 72 h,the expression of TNF-? and IL-6 in cell supernatants were detected by ELISA.Secondly,HMC-1 was stimulated with PBS or 100 nM CRH for 12,24,48 and 72 h,then the culture medium of HMC-1 were taken for incubating microglia for 24 h,ELISA was used to detect the expression of TNF-? and IL-6 in culture medium of microglia.Finally,microglia and HMC-1 were mixed directly for co-cultured,then exposed to 100 nM CRH or control PBS for 12,24,48 and 72 h,ELISA was used to detect the expression of TNF-? and IL-6 in culture medium.4.The microglia was pretreated with antagonist of H₁R,H₂R,H₃R,H₄R,PAR2,neurokinin 1(NK-1)and TLR4 receptors for 30 min,then exposed to culture medium of CRH-stimulated HMC-1 for 24 h,ELISA was used to detect the expression of TNF-? and IL-6 in culture medium.5.Firstly,HMC-1 was stimulated 100 nM CRH for 48 h,then the culture medium of HMC-1 were taken for incubating microglia for 15,30,60,120 and 240 min,Western blot was used to detect the phosphorylation of AKT and MAPK.Then,The microglia was pretreated with antagonist of H₁R,H₂R,H₃R,H₄R,PAR2,neurokinin 1(NK-1)and TLR4 receptors for 30 min,then exposed to culture medium of CRH-stimulated HMC-1,Western blot was used to detect the phosphorylation of AKT and MAPK.Results:1.Stimulation of HMC-1 with CRH caused a dose-dependent(1-104 nM)andtime-dependent(>24h)HMC-1 activation,indicated by the increase of Fc-positive cells number.However,treatment of CRH(1-104 nM)for 12h had no effect on HMC-1 activation.Incubation with CRH(100 nM)for more than 48 h,HMC-1 produced TNF-? and IL-6.2.Microglia was activated by CM from CRH-stimulated HMC-1 cells for 48 and 72 h,respectively.3.Incubation with CRH(100 nM)for 12,24,48 and 72 h could not increase the production of TNF-? and IL-6,but decrease the production of TNF-? and IL-6 for 48 and 72 h.The CM from CRH-stimulated HMC-1(48 and 72 h)induced the release of TNF-? and IL-6 from microglia following 24 h incubation period.Co-culture of microglia and HMC-1 cells with CRH(100 nM)for 24,48 and 72 h increased the production of TNF-? and IL-6 compare to culture of microglia alone.4.The antagonists of H₁R,H₄R,PAR2 and TollR4 can partially abolished CM from CRH-stimulated HMC-1 induced TNF-? and IL-6 releases from microglia.5.Treatment with CM from CRH-stimulated HMC-1 cells(48h)led to a rapid and transient phosphorylation of ERK,p38,JNK and AKT,indicative of ERK,p38,JNK and AKT activation,with the peak levels of phospho-ERK occurring at 60 min,phospho-p38 occurring at 15 min,phospho-JNK occurring at 120 min and phospho-AKT occurring at 120 min.The H₁R and H₄R antagonists suppressed JNK,p38 and AKT phosphorylation,and the PAR2 antagonist suppressed ERK,p38 and AKT phosphorylation in microglia.Conclusion:HMC-1 degranulation activate microglial activation via H₁R,H₄R,PAR2 and TLR4 receptor,and the activation of MAPK and AKT signaling pathways were involved.