Neuronal oxidative damage and dendritic degeneration following activation of CD14-dependent innate immune response in vivo
© Milatovic et al; licensee BioMed Central Ltd. 2004
Received: 6 October 2004
Accepted: 21 October 2004
Published: 21 October 2004
The cause-and-effect relationship between innate immune activation and neurodegeneration has been difficult to prove in complex animal models and patients. Here we review findings from a model of direct innate immune activation via CD14 stimulation using intracerebroventricular injection of lipopolysaccharide. These data show that CD14-dependent innate immune activation in cerebrum leads to the closely linked outcomes of neuronal membrane oxidative damage and dendritic degeneration. Both forms of neuronal damage could be blocked by ibuprofen and alpha-tocopherol, but not naproxen or gamma-tocopherol, at pharmacologically relevant concentrations. This model provides a convenient method to determine effective agents and their appropriate dose ranges for protecting neurons from CD14-activated innate immunity-mediated damage, and can guide drug development for diseases, such as Alzheimer disease, that are thought to derive in part from CD14-activated innate immune response.
Activated innate immunity is associated with several degenerative and destructive brain diseases including Alzheimer disease (AD), HIV-associated dementia (HAD), ischemia, head trauma, stroke, cerebral palsy, and axonal degeneration in multiple sclerosis . In this complex response, some aspects are proposed to be neurotrophic, others neurotoxic, and each potentially a consequence rather than a contributor to neurodegeneration. Indeed, a severe limitation to understanding the precise role of innate immunity in these diseases and their corresponding animal models is that innate immunity is activated simultaneously with multiple other stressors and responses to injury, thereby greatly confounding any clear conclusion about cause-and-effect relationships. For these reasons we have adopted a simple but highly specific model of isolated innate immune activation: intracerebroventricular (ICV) injection of low dose lipopolysaccharide (LPS).
LPS specifically activates innate immunity in peripheral organs through a well-described Toll-like receptor (TLR)-dependent signaling pathway [2, 3]. There are 9 known human plasma membrane-spanning TLRs expressed in many cell types throughout the body that have been discovered in the context of innate immune response to micro-organisms. TLR-mediated innate immune response can be considered in three phases: initial signal transduction cascade, secondary signaling cascades, and effectors. The initial signaling cascade starts with ligand activating one of the 9 plasma membrane TLRs. All of these receptors require the adaptor protein MyD88 for immediate response to LPS and initiate a bifurcated signal transduction cascade that culminates in altered gene transcription, primarily via NF-κB activation but also through c-Fos/c-Jun-dependent pathways. Some of the activated gene transcripts encode directly for receptor ligands while others are enzymes that catalyze the formation of receptor ligands that in turn activate secondary autocrine and paracrine signaling cascades. These signaling events culminate in the generation of effector molecules including bacteriocidal molecules, primarily free radicals generated by NADPH oxidase and myeloperoxidase (MPO), as well as cytokines and chemokines that can attract an adaptive immune response. Although originally identified as part of the response to exogenous antigens from micro-organisms, a broader pathophysiologic role for TLR-dependent signaling in response to endogenous ligands in now clear. Indeed, from this perspective, the effectors at the culmination of these signaling pathways are more appropriately viewed as cytocidal rather than specifically bacteriocidal. The precise agents responsible for cytocidal activity are not clearly established but likely include free radicals generated principally by NADPH oxidase, MPO, and inducible nitric oxide synthase (iNOS) in combination with cytokines and chemokines.
TLR-4 is the receptor for LPS in peripheral organs [2, 3]. However, another protein, CD14 is critical to LPS activation of TLR-4. Membrane-anchored CD14 is now thought to act a co-receptor for LPS but not to initiate intracellular signaling cascades. It is important to note that CD14 serves a similar function with TLR-2, although the activating agents here are bacterial products other than LPS . Within minutes to hours of exposure to LPS, there is increased gene transcription and subsequent translation of cytokines and chemokines, prominently including tumor necrosis factor, interleukin-1, and interferons, as well as several enzymes; important among these are iNOS and cyclooxygenase 2 (COX-2) that catalyze the formation of NO and prostaglandin (PG) H2, respectively . While NO is a potent cell signaling molecule, PGH2 has relatively low receptor binding affinity but is rapidly and efficiently converted to multiple PGs or thromboxane A2, each of which are potent activators of a large family of G protein-coupled receptors . The combination of these initial and secondary signaling cascades produces a robust innate immune response. This same response can occur in response to endogenous ligands that also activate the CD14/TLR-4 pathway [2, 3]. Indeed, several endogenous CD14/TLR ligands have received increasing attention for their potential roles in human diseases , and polymorphisms in TLR-4 are associated with risk for atherosclerosis and asthma, as well as other human diseases . With respect to AD, amyloid beta (A ) fibrils have been shown to activate the microglial innate immune response through CD14-dependent mechanisms . Relevant to a broader range of neurodegenerative diseases, novel peptides and neoantigens exposed by apoptotic cells  also activate CD14-dependent innate immune response in macrophages. While none of these data point to CD14 or innate immune response as etiological in neurodegenerative disorders, these findings from in vitro and cell culture experiments raise the possibility that CD14-dependent signaling may be a common process shared in the pathogenesis of neurodegenerative diseases, especially AD.
Here we present our results from studies that have identified the molecular and pharmacologic determinants of ICV LPS-initiated cerebral neuronal damage in vivo. It is important to stress that several laboratories have shown that glia, predominantly microglia, are activated by LPS but that neurons do not respond to LPS because they lack the appropriate receptors [10, 11]. We measured two main endpoints; one biochemical and one structural. Since free radicals are a primary mechanism of cytocidal activity from innate immune response, we used a stable isotope dilution method with gas chromatography and negative ion chemical ionization mass spectrometry to quantify compounds formed by free radical attack on the neuronal membrane-enriched fatty acid, docosohexaenoic acid (DHA); we have termed these molecules F4-neuroprostanes (F4-NeuroPs) . In addition to this biochemical marker of neuronal oxidative damage, we directly quantified neuron number as well as dendrite length and spine density in pyramidal neurons of hippocampal sector CA1 using the Golgi impregnation technique followed by quantitative morphometry with Neurolucida (MicroBrightField, VT) .
Lack of adaptive immune response, fever, or structural damage to brain following ICV LPS
Neuronal oxidative damage
We first determined the time course of F4-NeuroP accumulation in cerebrum of wt mice exposed to ICV LPS and observed a delayed, transient elevation that peaks at approximately 24 hr after exposure and then returns to baseline by 72 hr post exposure . It is important to note that while detectable neuronal oxidative damage is delayed several hours following ICV LPS, others have shown that altered gene transcription and increased cytokine secretion occur rapidly and peak within a few hours of LPS exposure. As with oxidation of lipoproteins, it is likely that this delay in neuronal oxidative damage is related, at least in part, to the time required to deplete anti-oxidant defenses. Thus, despite the lack of tissue damage, adaptive immune cell infiltrate, or detectable neuron loss, there is significant, reversible free radical damage to neuronal membranes following ICV LPS.
Neuronal oxidative damage and dendritic degeneration in various knockout mice. Effects of ICV LPS treatment determined at 24 hr in mice homozygous deficient (knockout) for different genes or wildtype (wt) mice all on the C57Bl/6 genetic background (*P < 0.001 by Bonferroni-corrected repeated pair comparisons with ICV saline-exposed mice).
F 4 -NeuroPs
352 + 53*
32 + 4*
37 + 6*
87 + 14
101 + 8
92 + 11
37 + 5*
51 + 8*
98 + 10
96 + 9
102 + 7
Initial Signal Cascade
108 + 11
105 + 7
106 + 10
92 + 12
103 + 8
97 + 6
89 + 9
102 + 12
109 + 5
These data left us with an apparent conflict. We have clearly demonstrated neuronal oxidative damage to mouse cerebrum following ICV LPS that is of a magnitude comparable to diseased regions of AD brain . However, there is no apparent structural damage to brain in our study or in others' following ICV or intraparenchymal LPS. We viewed this as a serious potential challenge to the significance of oxidative damage in neurodegeneration. There are differences, of course, between the acute stress of ICV LPS stress and the presumably chronic stress of AD; nevertheless, these data force at least consideration of the question: could oxidative damage to neurons occur in vivo to the extent that is observed in AD brain without any neurodegeneration?
Next, we extended our studies to tocopherols, natural antioxidant products with a number of proposed actions  including both anti-oxidant and anti-inflammatory activities . As with NSAIDs, α-tocopherol (AT) or γ-tocopherol (GT) alone does not alter basal F4-NeuroP levels or dendritie arbor (not shown). AT partially suppresses ICV LPS-induced F4-NeuroPs at 10 mg/kg and completely suppresses F4-NeuroP formation and both reduction in dendrite length and reduction in spine density at 100 mg/kg (Figure 5). GT, an isomer of AT that has one-tenth its anti-oxidant activity in vitro and lacks a specific transporter in vivo, does not, as expected, protect from neuronal oxidative damage or dendritic degeneration at the same dose.
Our data show that CD14-dependent activation of cerebral innate immunity leads to an acute, transient increase in oxidative damage to neuronal membranes that coincides with reversible dendritic degeneration. Although we did not directly test TLR-4 deficient mice in our studies, given what is know about LPS receptor activation and the fact that TLR-2-/- mice were not protected from neuronal damage caused by ICV LPS, these data argue strongly for CD14/TLR-4-dependent neuronal damage in our model. Moreover, using a wide array of genetically altered mice, we observed complete concordance between dendritic degeneration and neuronal membrane oxidative damage. In combination, these data suggest that these two events are mechanistically related, perhaps with neuronal membrane oxidative damage being a proximate contributor to dendritic degeneration in the context of innate immune activation.
One obvious, commonly voiced criticism of the model described here is that it produces an acute stress that does not correspond to chronic neurodegenerative diseases. However, it has yet to be shown whether the stress to individual neurons in these protracted diseases truly is chronic or instead the integration of innumerable microscopic acute stresses over many years. Finally, to the extent that CD14-dependent innate immunity activation contributes to neurodegenerative diseases, such as AD and HAD, the model described here provides a convenient means to screen experimental therapeutics and rapidly optimize dosing and timing parameters before moving to more complex animal models or clinical trials.
List of abbreviations used
prostaglandin E2 receptor subtype 2
inducible nitric oxide synthase
nonsteroidal anti-inflammatory drugs
This work was supported by the Alvord Endowed Chair in Neuropathology as well as grants from the NIH including AG05144, AG05136, and AG24011.
- Polazzi E, Contestabile A: Reciprocal interactions between microglia and neurons: from survival to neuropathology. Rev Neurosci. 2002, 13: 221 -2242.View ArticlePubMedGoogle Scholar
- Imler JL, Hoffmann JA: Toll receptors in innate immunity. Trends Cell Biol. 2001, 11: 304-311. 10.1016/S0962-8924(01)02004-9.View ArticlePubMedGoogle Scholar
- Akira S: Toll-like receptor signaling. J Biol Chem. 2003, 278: 38105-38108. 10.1074/jbc.R300028200.View ArticlePubMedGoogle Scholar
- Palsson-McDermott EM, O'Neill LA: Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology. 2004, 113: 153-162. 10.1111/j.1365-2567.2004.01976.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Hata AN, Breyer RM: Pharmacology and signaling of prostaglandin receptors: Multiple roles in inflammation and immune modulation. Pharmacol Ther. 2004, 103: 147-166. 10.1016/j.pharmthera.2004.06.003.View ArticlePubMedGoogle Scholar
- Johnson GB, Brunn GJ, Platt JL: Activation of mammalian Toll-like receptors by endogenous agonists. Crit Rev Immunol. 2003, 23: 15-44. 10.1615/CritRevImmunol.v23.i12.20.View ArticlePubMedGoogle Scholar
- Cook DN, Pisetsky DS, Schwartz DA: Toll-like receptors in the pathogenesis of human disease. Nat Immunol. 2004, 5: 975-979. 10.1038/ni1116.View ArticlePubMedGoogle Scholar
- Fassbender K, Walter S, Kuhl S, Landmann R, Ishii K, Bertsch T, Stalder AK, Muehlhauser F, Liu Y, Ulmer AJ, Rivest S, Lentschat A, Gulbins E, Jucker M, Staufenbiel M, Brechtel K, Walter J, Multhaup G, Penke B, Adachi Y, Hartmann T, Beyreuther K: The LPS receptor (CD14) links innate immunity with Alzheimer's disease. FASEB J. 2004, 18: 203-205.PubMedGoogle Scholar
- Moffatt OD, Devitt A, Bell ED, Simmons DL, Gregory CD: Macrophage recognition of ICAM-3 on apoptotic leukocytes. J Immunol. 1999, 162: 6800-6810.PubMedGoogle Scholar
- Minghetti L, Levi G: Induction of prostanoid biosynthesis by bacterial lipopolysaccharide and isoproterenol in rat microglial cultures. J Neurochem. 1995, 65: 2690-2698.View ArticlePubMedGoogle Scholar
- Fiebich BL, Schleicher S, Spleiss O, Czygan M, Hull M: Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C. J Neurochem. 2001, 79: 950-958. 10.1046/j.1471-4159.2001.00652.x.View ArticlePubMedGoogle Scholar
- Roberts L J, 2nd, Montine TJ, Markesbery WR, Tapper AR, Hardy P, Chemtob S, Dettbarn WD, Morrow JD: Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid. J Biol Chem. 1998, 273: 13605 -136012. 10.1074/jbc.273.22.13605.View ArticlePubMedGoogle Scholar
- Leuner B, Falduto J, Shors TJ: Associative memory formation increases the observation of dendritic spines in the hippocampus. J Neurosci. 2003, 23: 659 -6665.PubMed CentralPubMedGoogle Scholar
- Milatovic D, Zaja-Milatovic S, Montine KS, Horner PJ, Montine TJ: Pharmacologic suppression of neuronal oxidative damage and dendritic degeneration following direct activation of glial innate immunity in mouse cerebrum. J Neurochem. 2003, 87: 1518-1526.View ArticlePubMedGoogle Scholar
- Stern EL, Quan N, Proescholdt MG, Herkenham M: Spatiotemporal induction patterns of cytokine and related immune signal molecule mRNAs in response to intrastriatal injection of lipopolysaccharide. J Neuroimmunol. 2000, 106: 114-129. 10.1016/S0165-5728(00)00194-6.View ArticlePubMedGoogle Scholar
- Montine TJ, Milatovic D, Gupta RC, Valyi-Nagy T, Morrow JD, Breyer RM: Neuronal oxidative damage from activated innate immunity is EP2 receptor-dependent. J Neurochem. 2002, 83: 463-470. 10.1046/j.1471-4159.2002.01157.x.View ArticlePubMedGoogle Scholar
- Nadeau S, Rivest S: Endotoxemia prevents the cerebral inflammatory wave induced by intraparenchymal lipopolysaccharide injection: role of glucocorticoids and CD14. J Immunol. 2002, 169: 3370-3381.View ArticlePubMedGoogle Scholar
- Nadeau S, Rivest S: Glucocorticoids play a fundamental role in protecting the brain during innate immune response. J Neurosci. 2003, 23: 5536-5544.PubMedGoogle Scholar
- Montine TJ, Neely MD, Quinn JF, Beal MF, Markesbery WR, Roberts L J, 2nd, Morrow JD: Lipid peroxidation in aging brain and Alzheimer's disease. Free Radic Biol Med. 2002, 33: 620-626. 10.1016/S0891-5849(02)00807-9.View ArticlePubMedGoogle Scholar
- Morrow JD, Roberts LJ: The isoprostanes: unique bioactive products of lipid peroxidation. Prog Lipid Res. 1997, 36: 1 -21. 10.1016/S0163-7827(97)00001-5.View ArticlePubMedGoogle Scholar
- Montine KS, Quinn JF, Zhang J, Fessel JP, Roberts L J, 2nd, Morrow JD, Montine TJ: Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases. Chem Phys Lipids. 2004, 128: 117-124. 10.1016/j.chemphyslip.2003.10.010.View ArticlePubMedGoogle Scholar
- Liu B, Gao HM, Wang JY, Jeohn GH, Cooper CL, Hong JS: Role of nitric oxide in inflammation-mediated neurodegeneration. Ann NY Acad Sci. 2002, 962: 318-331.View ArticlePubMedGoogle Scholar
- Xie Z, Wei M, Morgan TE, Fabrizio P, Han D, Finch CE, Longo VD: Peroxynitrite mediates neurotoxicity of amyloid beta-peptide1-42- and lipopolysaccharide-activated microglia. J Neurosci. 2002, 22: 3484-3492.PubMedGoogle Scholar
- Rivest S: Molecular insights on the cerebral innate immune system. Brain Behav Immun. 2003, 17: 13 -119. 10.1016/S0889-1591(02)00055-7.View ArticlePubMedGoogle Scholar
- Beschorner R, Nguyen TD, Gozalan F, Pedal I, Mattern R, Schluesener HJ, Meyermann R, Schwab JM: CD14 expression by activated parenchymal microglia/macrophages and infiltrating monocytes following human traumatic brain injury. Acta Neuropathol (Berl). 2002, 103: 541-549. 10.1007/s00401-001-0503-7.View ArticleGoogle Scholar
- Lehnardt S, Massillon L, Follett P, Jensen FE, Ratan R, Rosenberg PA, Volpe JJ, Vartanian T: Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc Natl Acad Sci U S A. 2003, 100: 8514-8519. 10.1073/pnas.1432609100.PubMed CentralView ArticlePubMedGoogle Scholar
- Sugimoto Y, Shigemoto R, Namba T, Negishi M, Mizuno N, Narumiya S, Ichikawa A: Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 in the mouse nervous system. Neuroscience. 1994, 62: 919 -9928. 10.1016/0306-4522(94)90483-9.View ArticlePubMedGoogle Scholar
- Dumont I, Peri KG, Hardy P, Hou X, Martinez-Bermudez AK, Molotchnikoff S, Varma DR, Chemtob S: PGE2, via EP3 receptors, regulates brain nitric oxide synthase in the perinatal period. Am J Physiol. 1998, 275: R1812 - R1821.PubMedGoogle Scholar
- Zhang J, Rivest S: Distribution, regulation and colocalization of the genes encoding EP2 and EP4 PGE2 receptors in the rat brain and neuronal responses to inflammation. Eur J Neurosci. 1999, 11: 2651 -22668. 10.1046/j.1460-9568.1999.00682.x.View ArticlePubMedGoogle Scholar
- Ek M, Arias C, Sawchenko P, Ericsson-Dahlstrand A: Distribution of the EP3 prostaglandin E2 receptor subtype in the rat brain: relationship to sites of interleukin-1-induced cellular responsiveness. J Comp Neurol. 2000, 428: 5 -20. 10.1002/1096-9861(20001204)428:1<5::AID-CNE2>3.0.CO;2-M.View ArticlePubMedGoogle Scholar
- Nakamura K, Kaneko T, Yamashita Y, Hasegawa H, Katoh H, Negishi M: Immunohistochemical localization of prostaglandin EP3 receptor in the rat central nervous system. J Comp Neurol. 2000, 421: 543 -5569. 10.1002/(SICI)1096-9861(20000612)421:4<543::AID-CNE6>3.0.CO;2-3.View ArticlePubMedGoogle Scholar
- McCullough L, Wu L, Haughey N, Liang X, Hand T, Wang Q, Breyer RM, Andreasson K: Neuroprotective function of the PGE2 EP2 receptor in cerebral ischemia. J Neurosci. 2004, 24: 257-268. 10.1523/JNEUROSCI.4485-03.2004.View ArticlePubMedGoogle Scholar
- Reich EE, Markesbery WR, Roberts L J, 2nd, Swift LL,, Morrow JD, Montine TJ: Brain regional quantification of F-ring and D/E-ring isoprostanes and neuroprostanes in Alzheimer's disease. Am J Pathol. 2001, 158: 293 -2937.PubMed CentralView ArticlePubMedGoogle Scholar
- Weggen S, Eriksen JL, Das P, Sagi SA, Wang R, Pietzik CU, Findlay KA, Smith TE, Murphy MP, Butler T, Kang DE, Sterling N, Golde TE, Koo EH: A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature. 2001, 414: 212 -2216. 10.1038/35102591.View ArticlePubMedGoogle Scholar
- Brigelius-Flohe R, Traber MG: Vitamin E: function and metabolism. FASEB J. 1999, 13: 1145-1155.PubMedGoogle Scholar
- Li Y, Liu L, Barger SW, Mrak RE, Griffin WS: Vitamin E suppression of microglial activation is neuroprotective. J Neurosci Res. 2001, 66: 163-170. 10.1002/jnr.1208.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.