Histamine Regulates Astrocytes Immune Responses

Background:Postoperative cognitive dysfunction?POCD?is a common complication of central nervous system?CNS?in aged patients.Neuroinflammation induced by peripheral trauma plays a key role in the development of POCD.Mast cells?MCs?have been demonstrated to contribute to postoperative neuroinflammation in our previous studies.Under various types of stress,MCs serve as important sources of several mediators,including TNF-?,tryptase,and histamine.Histamine is a potent mediator of inflammation and a regulator of innate and acquired immunity.We have previously reported that microglia express histamine H1,H2,H3,and H4 Receptors,and histamine can induce microglia activation and inflammatory mediator release.Astrocytes have been conceptualized as an inert scaffold or as housekeeping cells for many years.However,a growing body of evidence suggest that this cell population actively modulates immune responses in the CNS.Mild activation of astrocytes usually exerts neuroprotective effects and ameliorates early symptoms of neurodegeneration.For instance,the release of neurotrophic factors such as glial cell-derived neurotrophic factor?GDNF?,brain-derived neurotrophic factor?BDNF?,and neurotrophin-3?NT-3?can promote neuronal survival and maintain synaptic homeostasis.In particular,recent studies suggest that GDNF can also inhibit microglial activation and alleviate neuroinflammation.However,strong activation of astrocytes leads to the secretion of large amounts of cytokines,chemokines,reactive oxygen species,and proinflammatory mediators,leading to excitotoxicity,neurodegeneration,and apoptosis.Astrocytes are the most abundant non-neuronal cell population in the CNS.However,few data are available regarding the interactions of histamine with astrocyte immunomodulatory function.Objective:In the present study,we investigated the expression of histamine receptors on astrocytes and the mechanism of the histamine-induced neuroprotective effect of astrocytes.Methods:1.Immunofluorescence,Western blot,and real-time PCR were used to detect the expression of four histamine receptors on astrocytes.2.Astrocytes were exposed to different doses of histamine?0.001–1?g/ml?for 24 h. Immunofluorescence and Western blot were used to detect the expression of GFAP.Given the notable increase of astrocyte GFAP expression in response to histamine challenge?0.1?g/ml?,we concurrently evaluated the expression of histamine receptors using Western blot,immunofluorescence and real-time PCR.3.Astrocytes were incubated with various concentration of histamine?0.001–1?g/ml?for 24 h.Or,Astrocytes were treated with 0.1?g/ml histamine for 2,6,12,24,48,and 72 h,respectively.ELISA was used to detect the production of TNF-? and IL-1?in culture medium.Based on the results of the step above,Astrocytes were pretreated with 10?M antagonist of three histamine receptors for 30 min and then exposed to histamine?0.1?g/ml?for 24h.the cell suspensions were collected for ELISA.4.Based on the observation above,Astrocytes were exposed to histamine at 0.01 and 0.1?g/ml for 6 and 24h.Or,Astrocytes were pretreated with 10?M antagonist of three histamine receptors for 30 min before being challenged with histamine?0.1?g/ml?for 24 h.Immunofluorescence and Western blot were used to detect the expression of GDNF.Results:1.Immunofluorescence,Western blot,and real-time PCR confirmed that primary rat astrocytes express histamine H₁,H₂,and H₃,but not H₄ receptors.2.Histamine could upregulate the expression of GFAP on astrocytes in a dose-dependent manner;histamine?0.1?g/ml?provoked significant upregulation of the expression of H₁R and H₃R in astrocytes.Meanwhile,no obvious increase in H₂R expression was found.3.Histamine could suppress the production of TNF-?and IL-1?by astrocytes;the TNF-?and IL-1?decrease induced by histamine?0.1?g/ml?was reversed by the H₁R antagonist,the H₃R antagonist,and partly by the H₂R antagonist.4.Histamine markedly promoted GDNF secretion from astrocytes;the increase in GDNF expression induced by histamine?0.1?g/ml?was almost completely blocked by either the H₁R or the H₃R antagonist,while the H₂R antagonist partly attenuated the stimulatory effect of histamine.Conclusion:Astrocytes express histamine H₁,H₂,and H₃,but not H₄ receptors;negative regulation of astrocytic TNF-?and IL-1?production and the enhancement of astrocytic GDNF stimulated by histamine were receptor-mediated processes in which all three of the expressed histamine receptors?H₁R,H₂R,and H₃R?were involved.