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Inflammation, the complement system and the diseases of aging
Neurobiology of Aging 26S (2005) S94–S97
Inflammation, the complement system and the diseases of aging Edith G. McGeer, Andis Klegeris, Patrick L. McGeer ∗ Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 Received 27 July 2005; accepted 17 August 2005
Abstract Inflammation is characteristic of neurodegenerative diseases of aging. Neuropathological evidence of activated microglia and activated astrocytes in lesioned areas, combined with epidemiological evidence of sparing of Alzheimer’s disease (AD), Parkinson’s disease (PD) and age-related macular degeneration (AMD) in long-term users of anti-inflammatory agents, indicates that inflammation is autodestructive of neurons. Locally produced autodestructive molecules include the membrane attack complex (MAC) of complement and oxygen-free radicals. Stimulation is provided by a variety of inflammatory cytokines. Agents which reduce the intensity of inflammation should have broad spectrum application in degenerative diseases of aging. © 2005 Elsevier Inc. All rights reserved. Keywords: Macular degeneration; Alzheimer’s disease; Parkinson’s disease; Factor H; Activated microglia; Membrane attack complex; Cytokines; Complement; NSAIDs; Atherosclerosis
1. Introduction Chronic inflammation appears to be a complicating factor in many of the important diseases of aging. Immunohistochemical and the molecular genetic evidence of chronic inflammation in affected tissue is seen not only in Alzheimer’s disease (AD) [26], Parkinson’s disease (PD) [27], ALS [24] and multiple sclerosis [6], but also in other conditions, such as atherosclerosis [16], heart disease [33,39] and age-related macular degeneration (AMD) [3]. More than 20 epidemiological studies, indicating that the chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) greatly reduces the risk of AD, testify to the importance of the inflammatory reaction in AD. Recent single epidemiological studies indicate that such use of NSAIDs also reduces the risk of PD [8] and AMD [29]. Moreover, inheritance of polymorphisms of various inflammatory mediators which enhance their expression have been reported to increase the risk of AD [26] and PD [27]. Identification of key mediators of the inflammatory reaction and development of effective inhibitors would seem to deserve very high priority. This brief ∗
Corresponding author. Tel.: +1 604 822 7377; fax: +1 604 822 7086. E-mail address: [email protected] (P.L. McGeer).
0197-4580/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2005.08.008
paper will focus on inflammation, the complement system and microglia.
2. Complement A prominent part of many neuroinflammatory reactions is the activation of complement. Complement [26] is a sophisticated attack system designed to destroy invaders, stimulate inflammation and assist in the phagocytosis of waste materials. The classical pathway is activated by the attachment of C1q to a target causing C1 dissociation. Amplification takes place through a cascade of proteases (C1r, C1s, C4, C2, C3), and the cleavage products C4b and C3b attach to the exposed sites close to the C1q binding site, opsonizing the target for phagocytosis. If the complement system is fully activated, it proceeds to assemble the terminal components (C5b, C6, C7, C8, C9) into the lytic macromolecule C5b-9, known as the membrane attack complex (MAC). The MAC is intended to insert itself into the foreign bacteria and viruses, but, if host cells are inadequately protected, it may damage them as well in a process called bystander lysis. Meanwhile, the small fragments C3a, C4a and C5a, known as anaphylotoxins, stimulate inflammation. So the overall cascade identifies,
E.G. McGeer et al. / Neurobiology of Aging 26S (2005) S94–S97
opsonizes and destroys its target, while dispatching messengers to seek help. The alternative complement pathway begins differently but also ends up with the formation of the MAC. The alternative pathway is independent of antibodies and other molecules which bind to C1q. It is activated by molecules, such as lipopolysaccharides found on the surface of bacteria and other foreign invaders. Specific activators bind to C3b which is continuously generated at very low levels in the serum. When binding occurs, there is a subsequent reaction with Factors B, D and P (properdin) to form a surfacebound enzymatic complex, C3bBb. This complex powerfully cleaves more C3, providing a strong feedback reaction. Regulatory molecules of the alternative pathway are Factor I, which attacks C3b, and Factor H which binds to C3b and accelerates the destructive action of Factor I on C3b. Polymorphisms in Factor H are known to increase or decrease the risk of AMD several fold [11,13,14,21]. This indicates that AMD involves the activation of the alternative pathway by unknown mechanisms, and that it is highly sensitive to the protection afforded by Factor H. Both pathways may be involved in various diseases. The classical pathway plays the major role in AD [26] with the alternative pathway also occurring [36]. The reverse holds true in AMD since Creactive protein (CRP), an activator of the classical pathway, appears to affect the risk much less than polymorphisms in Factor H [35]. Antibodies are considered to be the chief activators of the classical complement pathway. It has now become clear, however, that there can be vigorous activation of complement in the absence of antibodies. A key finding regarding the mechanism of complement activation in AD was that of Rogers et al. [34], who demonstrated that amyloid protein, when aggregated, was a strong complement activator. Thus, the senile plaques of AD have a unique activator of complement. In addition, the complement cascade can be activated by the pentraxins, amyloid P and CRP, which are both upregulated in affected regions of AD brain [26]. It is interesting that elevated levels of serum CRP are associated with both heart disease [33] and AMD [35].
3. Microglia A key cellular event signaling the presence of neuroinflammation is the accumulation of reactive microglia in the affected areas. Such activated microglia are seen after brain injury or in neurological diseases, such as AD or PD. Clumps of activated microglia appear on the senile plaques of AD, and many others are evident in the surrounding tissue [26]. Many activated microglia are also seen in the substantia nigra in PD [25] and in the spinal cord in ALS [17,18]. Few, if any, are seen in similar regions of control brains. Both aggregated beta-amyloid and ␣-synuclein are excellent activators of microglia, perhaps accounting for their appearance in AD and PD. Evidence from both humans [23] and monkeys [4,28]
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who received MPTP indicates that the microglia may remain chronically activated for years after the precipitating insult. These reactive microglia can be observed immunohistochemically using antibodies against markers which include, but are by no means restricted to, MHC class II glycoproteins, such as HLA-DR [26], beta-2-integrins, particularly the complement receptor CD1 1b [2], leukocyte common antigen (LCA) and the immunoglobulin receptor Fc␥RI. All of these are receptor proteins that are highly upregulated when cells of the monocyte lineage are activated, and immunohistochemistry for each reveals a similar cellular morphology. In the gray matter, the cells become hypertrophied to form the classic activated microglia described by Hortega [9] and Penfield [32]. They retain their processes which become thickened and retracted. In the white matter, the cells become engorged with myelin products, lose their processes, and become “fat granule cells” as originally described by Penfield. Various cytokines seem to be involved in signaling for activation. Cytokines are a heterogeneous group of small molecules that act in autocrine and paracrine fashion. They encompass several subfamilies which include interleukins (ILs), interferons (INFs), tumor necrosis factors (TNFs), growth factors, colony stimulating factors and chemokines. They have a common participation in inflammatory reactions. They typically act in combination so that attributing a specific set of in vivo properties to any given cytokine is difficult. IL-1␣, IL-1, IL-6, TNF␣ all seem to be involved in signaling for activation, whereas IL-4 signals for downregulation [26]. The importance of TNF␣ as an inflammatory stimulus is emphasized by the widespread use in rheumatoid arthritis of TNF␣ blockers [15].
4. Neurotoxicity The activation of the complement system and microglia in inflammation is intended as beneficial and part of the repair process. It is clear, however, that overactivation can lead to further damage and exacerbate disease processes. This has been clearly stated not only with regard to AD [26], but also in conditions, such as glaucoma. Thus, for example, Yuan and Neufeld [40] reported: “Our findings suggest that activated microglia may participate in stabilizing the tissue early in the disease process, but, as the severity of the glaucomatous damage increases, the activities of microglia may have detrimental consequences for the pathological course of glaucomatous optic neuropathy.” Just as microglia and the complement system are key elements in the beneficial processes, they appear to be equally important in neurotoxicity. The immunohistochemical demonstration of the MAC on dystrophic neurites in AD brain [26] indicates that overactivation of the complement system plays a neurotoxic role. Such staining is not seen in control brains. The MAC has a very short half-life so that the abundant staining for the MAC in AD brain suggests that such attack is responsible for much of the neurite loss in AD. The MAC has also been
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identified in affected regions in PD [27], ALS [24] and AMD [3]. A greater part of the autotoxic attack in AD, and possibly in other chronic neurological diseases, such as PD, is probably due to products secreted by the activated microglia. There is abundant in vitro and some in vivo evidence that such microglia are toxic to neurons [26]. Oxidative stress may be a particularly important factor. It had long been suspected that oxidative stress contributes to the lesions of AD and PD but the source of oxygen-free radicals was usually attributed to accidental leakage from the electron transport chain of neuronal mitochondria. However, the most abundant source of oxygen-free radicals in the CNS is the respiratory burst system of activated microglia. When the system is turned on, large numbers of superoxide ions are generated on the microglial external membrane, from where they are released into the surrounding as a purposeful attack system [26]. Other less abundant sources of free radicals are xanthine oxidase and nitric oxide synthase. Evidence of free radical attack in AD brain includes the presence of proteins that have been modified by glycation; the existence of low molecular weight compounds that have been oxidized and nitrated, such as 4-hydroxynonenal, malondialdehyde, 3-nitrotyrosine, 3-nitro-4-hydroxyphenylacetic acid, 5-nitrotocopherol and 8-hydroxy-deoxyguanosine; and the identification of lipids that have been peroxidated [7,26]. The nitrated products are mostly derived from peroxynitrite, a reaction product of nitric oxide and superoxide anions. The importance of oxidative attack by activated microglia has been recently emphasized by the work of Gao et al. [12] on the neurotoxicity of activated microglia towards dopaminergic neurons in models of PD. The respiratory burst depends upon the assembly of an NADPH-dependent enzyme on the membrane surface. Gao et al. have shown that peptide inhibitors of this enzyme markedly reduce the toxicity of activated microglia, and that neurotoxicity in the system is eliminated in NADPH knockout mice. Activated microglia are also a source of neurotoxic materials other than free radicals. Glutamate has been identified as one potential neurotoxin released by activated microglia but there are others of low molecular weight that have not been identified. It seems probable that a combination of materials is responsible for the observed neurotoxic effects [20].
5. Healing If an inflammatory problem is resolved, healing occurs, as seen, for example, after a kainic acid lesion to the cerebral cortex [1]. Microglia return to the inactivated state or undergo apoptosis. Again cytokine signaling agents are believed to be involved although the exact agents and processes are not yet known. We have observed that interleukin IL-4 is capable of downregulating human microglia neurotoxicity [19]. Other investigators have observed downregulation by this cytokine of several inflammatory markers and mediators,
including superoxide anion, IL-6, TNF␣, major histocompatibility complex class II molecules, intercellular adhesion molecule-1 (ICAM-1) and CD40 [5,37,38]. Therefore IL-4stimulated microglia show certain similarities to alternatively activated murine macrophages [30]. An anti-inflammatory phenotype of microglia may also be induced by ligands of phosphatidylserine receptors. It is believed that these receptors mediate phagocytosis of apoptotic cells by microglia, indicating that an inflammatory reaction is not required for the physiological removal of the damaged neurons (reviewed in [10]). In line with such a beneficial effect is the report that phosphatidylserine-containing liposomes have a protective effect against the decreases in IL-4 and impairment in long-term potentiation in aged rat hippocampus [31].
6. Conclusions There is considerable evidence that chronic inflammation plays an important role in the progression of a number of diseases of aging. Recent animal experiments suggest that factors such as diet may play a role since a high fat diet has been found to induce NADPH-associated oxidative stress and inflammation in rat cortex [41]. Whatever the contributing factors are, anti-inflammatory agents may slow the progression or retard the development of clinical disease. The NSAIDs currently used are inhibitors of minor players on the inflammatory stage. The MAC of complement and activated microglia appear to play a major role so that research aimed at developing effective inhibitors of these appears worthwhile. One possibility suggested by a recent article [22] is vaccinia virus complement control protein which is known to inhibit both complement pathways. It proved neuroprotective in a rat model of spinal cord injury.
Acknowledgments Our work has been supported by the Jack Brown and Family Alzheimer Disease Research Fund, the George Hodgson estate, and grants from the Pacific Parkinson’s Research Institute and the Alzheimer Society of Canada/CIHR/AstraZeneca.
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E.G. McGeer et al. / Neurobiology of Aging 26S (2005) S94–S97 [5] Benveniste EN, Nguyen VT, O’Keefe GM. Immunological aspects of microglia: relevance to Alzheimer’s disease. Neurochem Int 2001;39:381–91. [6] Bruck W, Stadelmann C. Inflammation and degeneration in multiple sclerosis. Neurol Sci 2003;24(Suppl. 5):S265–7. [7] Butterfield DA, Kanski J. Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins. Mech Aging Dev 2001;122:945–62. [8] Chen H, Zhang SM, Herman MA, Schwarzschild MA, Willett WC, Colditz GA, et al. Nonsteroidal anti-inflammatory drugs and the risk of Parkinson disease. Arch Neurol 2003;60:1059–64. [9] Del Rio Hortega P. El ‘tercer elemento’ de los centros nerviosos. Poder fagocitario y movilidad de la microglia. Bol Soc Esp Biol Ano 1919;ix:154–66. [10] De Simone R, Ajmone-Cat MA, Minghetti L. Atypical antiinflammatory activation of microglia induced by apoptotic neurons. Mol Neurobiol 2004;19:197–212. [11] Edwards AO, Ritter III R, Abel KJ, Manning A, Panhuysen C, Farrer LA. Complement Factor H polymorphism and age-related macular degeneration http://sciencexpress/www.sciencexpress.org/2005/ 10.1126/science.1110189. [12] Gao HM, Liu B, Hong JS. Critical role for microglial NADPH oxidase in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 2003;23:6181–7. [13] Hageman GS, Anderson DH, Johnson LV, Hancox LS, Talber AJ, Hardisty LI, et al. A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci USA 2005;102:7227–32. [14] Haines JL, Hauser MA, Schmidt S, Scott WK, Olson LM, Gallins P et al. Complement Factor H variant increases the risk of age-related macular degeneration. http://sciencexpress/www. sciencexpress.org/2005/10.1126/science.1110359. [15] Hamilton K, Clair EW. Tumour necrosis factor-alpha blockade: a new era for the effective management of rheumatoid arthritis. Expert Opin Pharmacother 2000;1:1041–52. [16] Hansson GK. Inflammation atherosclerosis and coronary artery disease. New Engl J Med 2005;352:1685–95. [17] Henkel JS, Engelhardt JI, Siklos L, Simpson EP, Kim SH, Pan T, et al. Presence of dendritic cells, MCP-1 and activated microglia/macrophages in amyotrophic lateral sclerosis spinal cord tissue. Ann Neurol 2004;55:221–35. [18] Kawamata T, Akiyama H, Yamada T, McGeer PL. Immunologic reactions in amyotrophic lateral sclerosis brain and spinal cord tissue. Am J Pathol 1992;140:691–707. [19] Klegeris A, Bissonnette CJ, McGeer PL. Modulation of human microglia and THP-1 cell toxicity by cytokines endogenous to the nervous system. Neurobiol Aging 2005;26:673–82. [20] Klegeris A, McGeer PL. Chymotrypsin-like proteases contribute to human monocyte THP-1 cell as well as human microglial neurotoxicity. Glia 2005;51:56–64. [21] Klein RJ, Zeiss C, Chew EY, Tsai J-Y, Sackler RS, Haynes C et al. Complement Factor H polymorphism in age-related macular degeneration. http://sciencexpress/www.sciencexpress.org/2005/ 10.1126/science.1109557. [22] Kulkarni AP, Kellaway LA, Lahiri DK, Kotwal GJ. Neuroprotection from complement-mediated inflammatory damage. Ann N Y Acad Sci 2004;1035:147–64.
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[23] Langston JW, Forno LS, Tetrud J, Reeves AG, Kaplan JA, Karluk D. Evidence of active nerve cell degeneration in the substantia nigra of humans years after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure. Ann Neurol 1999;46:598–605. [24] McGeer EG, McGeer PL. Pharmacologic approaches to the treatment of amyotrophic lateral sclerosis. Biodrugs 2005;19:31–7. [25] McGeer PL, Itagaki S, Boyes BE, McGeer EG. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology 1988;38:1285–91. [26] McGeer PL, McGeer EG. Inflammation and the degenerative diseases of aging. Ann N Y Acad Sci 2004;1035:101–4. [27] McGeer PL, McGeer EG. Inflammation and neurodegeneration in Parkinson’s disease. Parkinsonism Relat Disord 2004;10:S3–7. [28] McGeer PL, Schwab C, Parent A, Doudet D. Presence of reactive microglia in monkey substantia nigra years after 1-methyl4-phenyl-1,2,3,4-tetrahydropyridine administration. Ann Neurol 2003;54:599–604. [29] McGeer PL, Sibley J. Sparing of age-related macular degeneration in rheumatoid arthritis. Neurobiol Aging 2005;26:1199–203. [30] Mosser DM. The many faces of macrophage activation. J Leukoc Biol 2003;73:209–12. [31] Nolan Y, Martin D, Campbell VA, Lynch MA. Evidence of a protective effect of phosphatidyl-containing liposomes on lipopolysaccharide-induced impairment of long-term potentiation in the rat hippocampus. J Neurobiol 2004;151:12–23. [32] Penfield W. Microglia and the process of phagocytosis in gliomas. Am J Pathol 1925;1:77–89. [33] Ridker PM. C-reactive protein: a simple test to help predict risk of heart attack and stroke. Circulation 2003;108:81–5. [34] Rogers J, Webster S, Schultz J, McGeer PL, Styren S, Civin WH, et al. Complement activation by -amyloid in Alzheimer disease. Proc Natl Acad Sci USA 1992;89:10016–20. [35] Seddon JM, Gensler G, Milton RC, Klein ML, Rifai N. Association between C-reactive protein and age-related macular degeneration. J Am Med Assoc 2004;291:704–10. [36] Strohmeyer R, Shen Y, Rogers J. Detection of complement alternate pathway mRNA and proteins in the Alzheimer’s disease brain. Neurobiol Aging 2000;81:7–18. [37] Szczepanik AM, Funes S, Petko W, Ringheim GE. IL-4,IL-10 and IL-13 modulate A(1-42)-induced cytokine and chemokine production in primary murine microglia and a human monocyte cell line. J Neuroimmunol 2001;113:49–62. [38] Wirjatijasa F, Dehghani F, Blaheta RA, Korf HW, Hailer NP. Interleukin-4, interleukin-10, and interleukin-1-receptor antagonist but not transforming growth factor- induce ramification and reduce adhesion molecule expression of rat microglial cells. J Neurosci Res 2002;68:579–87. [39] Yasojima K, Schwab C, McGeer EG, McGeer PL. Human heart generates complement proteins that are upregulated and activated after myocardial infarction. Circ Res 1998;83:860–9. [40] Yuan L, Neufeld AH. Activated microglia in the human glaucomatous optic nerve head. J Neurosci Res 2001;64:523– 32. [41] Zhang X, Dong F, Ren J, Driscoll MJ, Culver B. High dietary fat induces NADPH oxidase-associated oxidative stress and inflammation in rat cerebral cortex. Exp Neurol 2005;191:318– 25.
Inflammation, the complement system and the diseases of aging Edith G. McGeer, Andis Klegeris, Patrick L. McGeer ∗ Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 Received 27 July 2005; accepted 17 August 2005
Abstract Inflammation is characteristic of neurodegenerative diseases of aging. Neuropathological evidence of activated microglia and activated astrocytes in lesioned areas, combined with epidemiological evidence of sparing of Alzheimer’s disease (AD), Parkinson’s disease (PD) and age-related macular degeneration (AMD) in long-term users of anti-inflammatory agents, indicates that inflammation is autodestructive of neurons. Locally produced autodestructive molecules include the membrane attack complex (MAC) of complement and oxygen-free radicals. Stimulation is provided by a variety of inflammatory cytokines. Agents which reduce the intensity of inflammation should have broad spectrum application in degenerative diseases of aging. © 2005 Elsevier Inc. All rights reserved. Keywords: Macular degeneration; Alzheimer’s disease; Parkinson’s disease; Factor H; Activated microglia; Membrane attack complex; Cytokines; Complement; NSAIDs; Atherosclerosis
1. Introduction Chronic inflammation appears to be a complicating factor in many of the important diseases of aging. Immunohistochemical and the molecular genetic evidence of chronic inflammation in affected tissue is seen not only in Alzheimer’s disease (AD) [26], Parkinson’s disease (PD) [27], ALS [24] and multiple sclerosis [6], but also in other conditions, such as atherosclerosis [16], heart disease [33,39] and age-related macular degeneration (AMD) [3]. More than 20 epidemiological studies, indicating that the chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) greatly reduces the risk of AD, testify to the importance of the inflammatory reaction in AD. Recent single epidemiological studies indicate that such use of NSAIDs also reduces the risk of PD [8] and AMD [29]. Moreover, inheritance of polymorphisms of various inflammatory mediators which enhance their expression have been reported to increase the risk of AD [26] and PD [27]. Identification of key mediators of the inflammatory reaction and development of effective inhibitors would seem to deserve very high priority. This brief ∗
Corresponding author. Tel.: +1 604 822 7377; fax: +1 604 822 7086. E-mail address: [email protected] (P.L. McGeer).
0197-4580/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2005.08.008
paper will focus on inflammation, the complement system and microglia.
2. Complement A prominent part of many neuroinflammatory reactions is the activation of complement. Complement [26] is a sophisticated attack system designed to destroy invaders, stimulate inflammation and assist in the phagocytosis of waste materials. The classical pathway is activated by the attachment of C1q to a target causing C1 dissociation. Amplification takes place through a cascade of proteases (C1r, C1s, C4, C2, C3), and the cleavage products C4b and C3b attach to the exposed sites close to the C1q binding site, opsonizing the target for phagocytosis. If the complement system is fully activated, it proceeds to assemble the terminal components (C5b, C6, C7, C8, C9) into the lytic macromolecule C5b-9, known as the membrane attack complex (MAC). The MAC is intended to insert itself into the foreign bacteria and viruses, but, if host cells are inadequately protected, it may damage them as well in a process called bystander lysis. Meanwhile, the small fragments C3a, C4a and C5a, known as anaphylotoxins, stimulate inflammation. So the overall cascade identifies,
E.G. McGeer et al. / Neurobiology of Aging 26S (2005) S94–S97
opsonizes and destroys its target, while dispatching messengers to seek help. The alternative complement pathway begins differently but also ends up with the formation of the MAC. The alternative pathway is independent of antibodies and other molecules which bind to C1q. It is activated by molecules, such as lipopolysaccharides found on the surface of bacteria and other foreign invaders. Specific activators bind to C3b which is continuously generated at very low levels in the serum. When binding occurs, there is a subsequent reaction with Factors B, D and P (properdin) to form a surfacebound enzymatic complex, C3bBb. This complex powerfully cleaves more C3, providing a strong feedback reaction. Regulatory molecules of the alternative pathway are Factor I, which attacks C3b, and Factor H which binds to C3b and accelerates the destructive action of Factor I on C3b. Polymorphisms in Factor H are known to increase or decrease the risk of AMD several fold [11,13,14,21]. This indicates that AMD involves the activation of the alternative pathway by unknown mechanisms, and that it is highly sensitive to the protection afforded by Factor H. Both pathways may be involved in various diseases. The classical pathway plays the major role in AD [26] with the alternative pathway also occurring [36]. The reverse holds true in AMD since Creactive protein (CRP), an activator of the classical pathway, appears to affect the risk much less than polymorphisms in Factor H [35]. Antibodies are considered to be the chief activators of the classical complement pathway. It has now become clear, however, that there can be vigorous activation of complement in the absence of antibodies. A key finding regarding the mechanism of complement activation in AD was that of Rogers et al. [34], who demonstrated that amyloid protein, when aggregated, was a strong complement activator. Thus, the senile plaques of AD have a unique activator of complement. In addition, the complement cascade can be activated by the pentraxins, amyloid P and CRP, which are both upregulated in affected regions of AD brain [26]. It is interesting that elevated levels of serum CRP are associated with both heart disease [33] and AMD [35].
3. Microglia A key cellular event signaling the presence of neuroinflammation is the accumulation of reactive microglia in the affected areas. Such activated microglia are seen after brain injury or in neurological diseases, such as AD or PD. Clumps of activated microglia appear on the senile plaques of AD, and many others are evident in the surrounding tissue [26]. Many activated microglia are also seen in the substantia nigra in PD [25] and in the spinal cord in ALS [17,18]. Few, if any, are seen in similar regions of control brains. Both aggregated beta-amyloid and ␣-synuclein are excellent activators of microglia, perhaps accounting for their appearance in AD and PD. Evidence from both humans [23] and monkeys [4,28]
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who received MPTP indicates that the microglia may remain chronically activated for years after the precipitating insult. These reactive microglia can be observed immunohistochemically using antibodies against markers which include, but are by no means restricted to, MHC class II glycoproteins, such as HLA-DR [26], beta-2-integrins, particularly the complement receptor CD1 1b [2], leukocyte common antigen (LCA) and the immunoglobulin receptor Fc␥RI. All of these are receptor proteins that are highly upregulated when cells of the monocyte lineage are activated, and immunohistochemistry for each reveals a similar cellular morphology. In the gray matter, the cells become hypertrophied to form the classic activated microglia described by Hortega [9] and Penfield [32]. They retain their processes which become thickened and retracted. In the white matter, the cells become engorged with myelin products, lose their processes, and become “fat granule cells” as originally described by Penfield. Various cytokines seem to be involved in signaling for activation. Cytokines are a heterogeneous group of small molecules that act in autocrine and paracrine fashion. They encompass several subfamilies which include interleukins (ILs), interferons (INFs), tumor necrosis factors (TNFs), growth factors, colony stimulating factors and chemokines. They have a common participation in inflammatory reactions. They typically act in combination so that attributing a specific set of in vivo properties to any given cytokine is difficult. IL-1␣, IL-1, IL-6, TNF␣ all seem to be involved in signaling for activation, whereas IL-4 signals for downregulation [26]. The importance of TNF␣ as an inflammatory stimulus is emphasized by the widespread use in rheumatoid arthritis of TNF␣ blockers [15].
4. Neurotoxicity The activation of the complement system and microglia in inflammation is intended as beneficial and part of the repair process. It is clear, however, that overactivation can lead to further damage and exacerbate disease processes. This has been clearly stated not only with regard to AD [26], but also in conditions, such as glaucoma. Thus, for example, Yuan and Neufeld [40] reported: “Our findings suggest that activated microglia may participate in stabilizing the tissue early in the disease process, but, as the severity of the glaucomatous damage increases, the activities of microglia may have detrimental consequences for the pathological course of glaucomatous optic neuropathy.” Just as microglia and the complement system are key elements in the beneficial processes, they appear to be equally important in neurotoxicity. The immunohistochemical demonstration of the MAC on dystrophic neurites in AD brain [26] indicates that overactivation of the complement system plays a neurotoxic role. Such staining is not seen in control brains. The MAC has a very short half-life so that the abundant staining for the MAC in AD brain suggests that such attack is responsible for much of the neurite loss in AD. The MAC has also been
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identified in affected regions in PD [27], ALS [24] and AMD [3]. A greater part of the autotoxic attack in AD, and possibly in other chronic neurological diseases, such as PD, is probably due to products secreted by the activated microglia. There is abundant in vitro and some in vivo evidence that such microglia are toxic to neurons [26]. Oxidative stress may be a particularly important factor. It had long been suspected that oxidative stress contributes to the lesions of AD and PD but the source of oxygen-free radicals was usually attributed to accidental leakage from the electron transport chain of neuronal mitochondria. However, the most abundant source of oxygen-free radicals in the CNS is the respiratory burst system of activated microglia. When the system is turned on, large numbers of superoxide ions are generated on the microglial external membrane, from where they are released into the surrounding as a purposeful attack system [26]. Other less abundant sources of free radicals are xanthine oxidase and nitric oxide synthase. Evidence of free radical attack in AD brain includes the presence of proteins that have been modified by glycation; the existence of low molecular weight compounds that have been oxidized and nitrated, such as 4-hydroxynonenal, malondialdehyde, 3-nitrotyrosine, 3-nitro-4-hydroxyphenylacetic acid, 5-nitrotocopherol and 8-hydroxy-deoxyguanosine; and the identification of lipids that have been peroxidated [7,26]. The nitrated products are mostly derived from peroxynitrite, a reaction product of nitric oxide and superoxide anions. The importance of oxidative attack by activated microglia has been recently emphasized by the work of Gao et al. [12] on the neurotoxicity of activated microglia towards dopaminergic neurons in models of PD. The respiratory burst depends upon the assembly of an NADPH-dependent enzyme on the membrane surface. Gao et al. have shown that peptide inhibitors of this enzyme markedly reduce the toxicity of activated microglia, and that neurotoxicity in the system is eliminated in NADPH knockout mice. Activated microglia are also a source of neurotoxic materials other than free radicals. Glutamate has been identified as one potential neurotoxin released by activated microglia but there are others of low molecular weight that have not been identified. It seems probable that a combination of materials is responsible for the observed neurotoxic effects [20].
5. Healing If an inflammatory problem is resolved, healing occurs, as seen, for example, after a kainic acid lesion to the cerebral cortex [1]. Microglia return to the inactivated state or undergo apoptosis. Again cytokine signaling agents are believed to be involved although the exact agents and processes are not yet known. We have observed that interleukin IL-4 is capable of downregulating human microglia neurotoxicity [19]. Other investigators have observed downregulation by this cytokine of several inflammatory markers and mediators,
including superoxide anion, IL-6, TNF␣, major histocompatibility complex class II molecules, intercellular adhesion molecule-1 (ICAM-1) and CD40 [5,37,38]. Therefore IL-4stimulated microglia show certain similarities to alternatively activated murine macrophages [30]. An anti-inflammatory phenotype of microglia may also be induced by ligands of phosphatidylserine receptors. It is believed that these receptors mediate phagocytosis of apoptotic cells by microglia, indicating that an inflammatory reaction is not required for the physiological removal of the damaged neurons (reviewed in [10]). In line with such a beneficial effect is the report that phosphatidylserine-containing liposomes have a protective effect against the decreases in IL-4 and impairment in long-term potentiation in aged rat hippocampus [31].
6. Conclusions There is considerable evidence that chronic inflammation plays an important role in the progression of a number of diseases of aging. Recent animal experiments suggest that factors such as diet may play a role since a high fat diet has been found to induce NADPH-associated oxidative stress and inflammation in rat cortex [41]. Whatever the contributing factors are, anti-inflammatory agents may slow the progression or retard the development of clinical disease. The NSAIDs currently used are inhibitors of minor players on the inflammatory stage. The MAC of complement and activated microglia appear to play a major role so that research aimed at developing effective inhibitors of these appears worthwhile. One possibility suggested by a recent article [22] is vaccinia virus complement control protein which is known to inhibit both complement pathways. It proved neuroprotective in a rat model of spinal cord injury.
Acknowledgments Our work has been supported by the Jack Brown and Family Alzheimer Disease Research Fund, the George Hodgson estate, and grants from the Pacific Parkinson’s Research Institute and the Alzheimer Society of Canada/CIHR/AstraZeneca.
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