Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and sPLA2-IIA expression in astrocytes and microglia

Journal of Neuroinflammation

Grace Y Sun⁶  Gary A Weisman²  Jau-Shyong Hong⁵  Zezong Gu⁶  Albert Y Sun⁶  Agnes Simonyi⁶  Jennifer L Hamilton²  Dongdong Han⁴  Jiankun Cui⁶  Deepa Ajit²  Arwa Mohammad¹  Yijia Zong³  Wenwen Sheng⁴ 

[1] Department of Biological Sciences, University of Missouri, Columbia, Missouri 65211, USA;Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211, USA;Interdisciplinary Neuroscience Program, University of Missouri, Columbia, Missouri 65211, USA;Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA;Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA;Center for Translational Neurosciences, University of Missouri Medical School, Columbia, Missouri 65212, USA

关键词: filopodia;    ERK1/2;    iNOS;    sPLA2-IIA;    primary microglial cells;    primary astrocytes;    DITNC;    HAPI;    BV-2;   

Others  :  1213111
DOI  :  10.1186/1742-2094-8-121

 received in 2011-07-01, accepted in 2011-09-24,  发布年份 2011

【 摘 要 】

Background

Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA₂-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes.

Methods/Results

Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA₂-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression.

Conclusion

These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.

【 授权许可】

   
2011 Sheng et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Benveniste EN, Nguyen VT, O'Keefe GM: Immunological aspects of microglia: relevance to Alzheimer's disease. Neurochem Int 2001, 39:381-391.
• [2]Yenari MA, Kauppinen TM, Swanson RA: Microglial activation in stroke: therapeutic targets. Neurotherapeutics 2011, 7:378-391.
• [3]Van Eldik LJ, Thompson WL, Ralay Ranaivo H, Behanna HA, Martin Watterson D: Glia proinflammatory cytokine upregulation as a therapeutic target for neurodegenerative diseases: function-based and target-based discovery approaches. Int Rev Neurobiol 2007, 82:277-296.
• [4]Miller DW, Cookson MR, Dickson DW: Glial cell inclusions and the pathogenesis of neurodegenerative diseases. Neuron Glia Biol 2004, 1:13-21.
• [5]Chen Y, Swanson RA: Astrocytes and brain injury. J Cereb Blood Flow Metab 2003, 23:137-149.
• [6]Schubert P, Ferroni S: Pathological glial reactions in neurodegenerative disorders: prospects for future therapeutics. Expert Rev Neurother 2003, 3:279-287.
• [7]Lu X, Ma L, Ruan L, Kong Y, Mou H, Zhang Z, Wang Z, Wang JM, Le Y: Resveratrol differentially modulates inflammatory responses of microglia and astrocytes. J Neuroinflammation 2010, 7:46. BioMed Central Full Text
• [8]Possel H, Noack H, Putzke J, Wolf G, Sies H: Selective upregulation of inducible nitric oxide synthase (iNOS) by lipopolysaccharide (LPS) and cytokines in microglia: in vitro and in vivo studies. Glia 2000, 32:51-59.
• [9]Shen S, Yu S, Binek J, Chalimoniuk M, Zhang X, Lo SC, Hannink M, Wu J, Fritsche K, Donato R, Sun GY: Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells. Neurochem Int 2005, 47:298-307.
• [10]Thampithak A, Jaisin Y, Meesarapee B, Chongthammakun S, Piyachaturawat P, Govitrapong P, Supavilai P, Sanvarinda Y: Transcriptional regulation of iNOS and COX-2 by a novel compound from Curcuma comosa in lipopolysaccharide-induced microglial activation. Neurosci Lett 2009, 462:171-175.
• [11]Jana M, Anderson JA, Saha RN, Liu X, Pahan K: Regulation of inducible nitric oxide synthase in proinflammatory cytokine-stimulated human primary astrocytes. Free Radic Biol Med 2005, 38:655-664.
• [12]Jung HW, Chung YS, Kim YS, Park YK: Celastrol inhibits production of nitric oxide and proinflammatory cytokines through MAPK signal transduction and NF-kappaB in LPS-stimulated BV-2 microglial cells. Exp Mol Med 2007, 39:715-721.
• [13]Murakami M, Taketomi Y, Girard C, Yamamoto K, Lambeau G: Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice. Biochimie 2010, 92:561-582.
• [14]Ibeas E, Fuentes L, Martin R, Hernandez M, Nieto ML: Secreted phospholipase A2 type IIA as a mediator connecting innate and adaptive immunity: new role in atherosclerosis. Cardiovasc Res 2009, 81:54-63.
• [15]Kimura-Matsumoto M, Ishikawa Y, Komiyama K, Tsuruta T, Murakami M, Masuda S, Akasaka Y, Ito K, Ishiguro S, Morita H, et al.: Expression of secretory phospholipase A2s in human atherosclerosis development. Atherosclerosis 2008, 196:81-91.
• [16]Lin TN, Wang Q, Simonyi A, Chen JJ, Cheung WM, He YY, Xu J, Sun AY, Hsu CY, Sun GY: Induction of secretory phospholipase A2 in reactive astrocytes in response to transient focal cerebral ischemia in the rat brain. J Neurochem 2004, 90:637-645.
• [17]Adibhatla RM, Hatcher JF: Secretory phospholipase A2 IIA is up-regulated by TNF-alpha and IL-1alpha/beta after transient focal cerebral ischemia in rat. Brain Res 2007, 1134:199-205.
• [18]Moses GS, Jensen MD, Lue LF, Walker DG, Sun AY, Simonyi A, Sun GY: Secretory PLA2-IIA: a new inflammatory factor for Alzheimer's disease. J Neuroinflammation 2006, 3:28. BioMed Central Full Text
• [19]Liu NK, Zhang YP, Titsworth WL, Jiang X, Han S, Lu PH, Shields CB, Xu XM: A novel role of phospholipase A2 in mediating spinal cord secondary injury. Ann Neurol 2006, 59:606-619.
• [20]Titsworth WL, Cheng X, Ke Y, Deng L, Burckardt KA, Pendleton C, Liu NK, Shao H, Cao QL, Xu XM: Differential expression of sPLA2 following spinal cord injury and a functional role for sPLA2-IIA in mediating oligodendrocyte death. Glia 2009, 57:1521-1537.
• [21]Jensen MD, Sheng W, Simonyi A, Johnson GS, Sun AY, Sun GY: Involvement of oxidative pathways in cytokine-induced secretory phospholipase A2-IIA in astrocytes. Neurochem Int 2009, 55:362-368.
• [22]Li W, Xia J, Sun GY: Cytokine induction of iNOS and sPLA2 in immortalized astrocytes (DITNC): response to genistein and pyrrolidine dithiocarbamate. J Interferon Cytokine Res 1999, 19:121-127.
• [23]Oka S, Arita H: Inflammatory factors stimulate expression of group II phospholipase A2 in rat cultured astrocytes. Two distinct pathways of the gene expression. J Biol Chem 1991, 266:9956-9960.
• [24]Kennedy BP, Payette P, Mudgett J, Vadas P, Pruzanski W, Kwan M, Tang C, Rancourt DE, Cromlish WA: A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains. J Biol Chem 1995, 270:22378-22385.
• [25]Cheepsunthorn P, Radov L, Menzies S, Reid J, Connor JR: Characterization of a novel brain-derived microglial cell line isolated from neonatal rat brain. Glia 2001, 35:53-62.
• [26]Horvath RJ, Nutile-McMenemy N, Alkaitis MS, Deleo JA: Differential migration, LPS-induced cytokine, chemokine, and NO expression in immortalized BV-2 and HAPI cell lines and primary microglial cultures. J Neurochem 2008, 107:557-569.
• [27]McCarthy KD, de Vellis J: Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980, 85:890-902.
• [28]Xu J, Chalimoniuk M, Shu Y, Simonyi A, Sun AY, Gonzalez FA, Weisman GA, Wood WG, Sun GY: Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents. Prostaglandins Leukot Essent Fatty Acids 2003, 69:437-448.
• [29]Li N, McLaren JE, Michael DR, Clement M, Fielding CA, Ramji DP: ERK is integral to the IFN-gamma-mediated activation of STAT1, the expression of key genes implicated in atherosclerosis, and the uptake of modified lipoproteins by human macrophages. J Immunol 2010, 185:3041-3048.
• [30]Chim SM, Qin A, Tickner J, Pavlos N, Davey T, Wang H, Guo Y, Zheng MH, Xu J: EGFL6 Promotes Endothelial Cell Migration and Angiogenesis through the Activation of Extracellular Signal-regulated Kinase. J Biol Chem 286:22035-22046.
• [31]Nakamura Y, Si QS, Kataoka K: Lipopolysaccharide-induced microglial activation in culture: temporal profiles of morphological change and release of cytokines and nitric oxide. Neurosci Res 1999, 35:95-100.
• [32]Chu SC, Marks-Konczalik J, Wu HP, Banks TC, Moss J: Analysis of the cytokine-stimulated human inducible nitric oxide synthase (iNOS) gene: characterization of differences between human and mouse iNOS promoters. Biochem Biophys Res Commun 1998, 248:871-878.
• [33]Jung JS, Kim DH, Kim HS: Ginsenoside Rh1 suppresses inducible nitric oxide synthase gene expression in IFN-gamma-stimulated microglia via modulation of JAK/STAT and ERK signaling pathways. Biochem Biophys Res Commun 2010, 397:323-328.
• [34]Kozuka N, Itofusa R, Kudo Y, Morita M: Lipopolysaccharide and proinflammatory cytokines require different astrocyte states to induce nitric oxide production. J Neurosci Res 2005, 82:717-728.
• [35]Saura J: Microglial cells in astroglial cultures: a cautionary note. J Neuroinflammation 2007, 4:26. BioMed Central Full Text
• [36]Wang JH, Sun GY: Platelet activating factor (PAF) antagonists on cytokine induction of iNOS and sPLA2 in immortalized astrocytes (DITNC). Neurochem Res 2000, 25:613-619.