The complement system in the peripheral nerve: Friend or foe?

Downloaded from UvA-DARE, the Institutional Repository of the University of Amsterdam (UvA) http://dare.uva.nl/document/115020 Description File ID Filename Chapter 1 Introduction 115020 04.pdf SOURCE, OR PART OF THE FOLLOWING SOURCE: Type Dissertation Title The complement system in the peripheral nerve Author V. Ramaglia Faculty Faculty of Medicine Year 2008 Pages 179 FULL BIBLIOGRAPHIC DETAILS: http://dare.uva.nl/record/282646 Copyrights It is not permitted to download or to forward/distribute the text or part of it without the consent of the copyright holder (usually the author), other then for strictly personal, individual use. UvA-DARE is a service provided by the Library of the University of Amsterdam (http://dare.uva.nl) Chapter 1 Introduction Ramaglia V. and F. Baas (2008) Innate immunity in the nervous system. An adapted form will be published as book chapter in Progress in Brain Research Contents he peripheral nerve Degeneration Regeneration Disease he complement system Origins of complement research A tight balance of activation and regulation Complement in inlammation Complement in inlammatory and neurodegenerative disorders Aim and outline of this thesis 10 The peripheral nerve 11 Introduction Figure 1. Schematic representation of the peripheral nervous system (in blue) in the human body. An enlargement of the peripheral nerve, showing its structure and various compartments, including the architecture of the axon and surrounding Schwann cell is shown. Modiied from: http://www.answers. com/topic/peripheral_nervous_system. Chapter 1 he peripheral nervous system (PNS) links the central nervous system (CNS) to the rest of the body. Peripheral nerve trunks consist of a varying number of fascicles containing axons, Schwann cells, ibroblasts and blood vessels, embedded in connective tissue called endoneurium and delimited by the perineurium. All fascicles lay in the epineurium. Axons are processes of neuronal cell bodies in the brain, spinal cord, autonomic and sensory ganglia. hey are either aferent or eferent with respect to the CNS, transmitting sensory or motor stimuli. Each axon is surrounded by many Schwann cells arranged at regular intervals along its length. Schwann cells wrap around the axon to form a cylindrical layer of lipids and proteins called myelin (Figure 1). Myelin provides physical protection for the axon and insulation to increase the conduction velocity of the electrical stimulus. he intervals between two Schwann cells (or myelin wraps) are structurally and molecularly highly organized regions called nodes of Ranvier. his architecture, which allows for the saltatory conduction of the electrical stimulus from one node of Ranvier to the next, is the result of an evolutionary adaptation to quickly perceive and respond to environmental stimuli. Figure 2. Drawing illustrating a group of 3 myelinated axons and the target skeletal muscle in the healthy nerve, in a nerve undergoing Wallerian degeneration and regeneration and in the regenerated nerve. Modiied from:http://missinglink.ucsf.edu/ lmlids_104_cns_injury/ Nerve bundles also contain unmyelinated axons. hese appear as groups of 1-8 smaller axons within the Schwann cell cytoplasm. hey are known as Remak ibers and have a sensory function 1. Degeneration Degeneration of the nerve after axotomy was irst described in 1850 by Augustus Waller 2 and it is since known as Wallerian degeneration (WD) (see scheme in igure 2). He transected the glossopharyngeal and hypoglossal nerves of the frog and described the observed process as “coagulation and curdling of the medulla into separate particles of various sizes” but it did not resolve the nature of the “medulla”. Fifty years later, Ramón y Cajal 3 conirmed Waller’s observations and determined that axonal degradation precedes the collapse of the myelin. He provided a very detailed histological description of axonal and myelin degeneration, Schwann cell responses and inlammatory iniltrates. he seminal observations of Waller and Cajal have been followed by a wealth of ultrastructural and molecular studies. Today the term Wallerian degeneration refers to the process of axonal degradation which occurs in the distal stump of an injured nerve following mechanical (i.e. transection, crush) or metabolic (i.e. ischemia, toxins) trauma. WD begins with axonal disintegration (Figure 3). his is mediated by calcium inlux and activation of calpains, calcium-dependant proteases 4. he initial changes are visible as early as 12 hours after injury and include the collection of organelle clumps which may represent a block of axonal transport. hese clumps appear mainly at nodes of Ranvier leaving the intercalating area void of axonal content. Approximately 2-3 days later, the irst changes in myelin structure occur. Myelin collapses into ellipsoids in the distal stump and in the most distal end of the proximal stump up to the irst intact node of Ranvier (Figure 3). At the same time, Schwann cells dediferentiate, multiply within their basal lamina tubes and downregulate myelin protein synthesis 5. hey 12 Chapter 1 Introduction Figure 3. Electron micrographs of the peripheral nerve showing the annulated myelin sheath containing the axoplasma in the uninjured nerve iber. At 1 day after a mechanical trauma, the degenerating nerve iber appears void of axonal content and at 3 days post-injury, the myelin has collapsed and appears as ellipsoids. he regenerative nerve iber shows a high number of clusters of small diameter and thinly myelinated axons. 13 break down the myelin ellipsoids into smaller ovoids and the myelin lipids eventually degenerate into neutral fat which is removed by macrophages. he endoneurial macrophage population consists of short-term and long-term resident macrophages 6. Starting at 2 days after injury, endoneurial macrophages proliferate, become activated and participate in myelin removal; however, they cannot efficiently complete myelin clearance. Monocyte/macrophages recruited to the injured site from the blood stream are responsible for the rapid and efficient clearance of myelin debris 7. he pathological changes of WD are well characterized but the molecular changes are poorly understood. he discovery of a mouse strain (ola, Wlds) with a very slow Wallerian degeneration 8 proved that axonal degradation is not a passive process due to lack of supply of newly synthesized proteins from the cell body but that it is an active and regulated “death program” similar to apoptosis. In Wlds, the gene encoding for the N-terminal 70 amino acids fragment of ubiquitination factor E4B (Ube4b) is fused to nicotinamide mononucleotide adenyltransferase-1 (Nmnat1), an essential enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD) 9. he resulting fusion protein has a protective efect on transected axons; namely, the distal stump of the transected axon and the neuromuscular junctions are protected from degeneration for a period of 2 weeks 10. he mechanism of Wlds protection of an injured axon remains elusive. he initial ideas of a dominant-negative efect of the ubiquitination factor fragment and/or the activity of Nmnat1 could not be conirmed. On one hand, nuclear localization of the Wlds protein does not support a putative proteasome malfunction in the axon 11. On the other hand, overexpression of Nmnat1 in transgenic mice is not sufficient to provide Wlds-like protection 12. he attention is now turned to the unique 18 amino acids linker region and the 3’ and 5’ untranslated sequences of the Wlds gene but the quest for the Wlds mode of action remains open. Interestingly, gene transfer of the Wlds protein delays axonal degeneration not only after mechanical injury but also after toxic challenge and in a variety of disorders including neuropathies 13. hese data show that WD is a process common to many injury and non-injury related disorders and imply that understanding the mechanism of WD could be the basis for therapy of neurodegenerative disorders and neuropathies. Regeneration Damaged axons of the CNS show little regenerative capability whereas peripheral axons often achieve a good morphological regeneration, but regain function slowly and incompletely 14,15. Peripheral nerve regeneration after injury requires axons to re-enter the Schwann cell tubes injured at the lesion site. he search of axons for the appropriate Schwann cell tube is represented by the axonal branches emerging from the tip of the proximal undamaged nerve stump. Once in the distal stump, the axons 14 Peripheral neuropathies are acquired or hereditary diseases of the peripheral nervous system. Acquired or inlammatory neuropathies, such as Guillain-Barré Syndrome (GBS) and its chronic form, chronic inlammatory demyelinating polyneuropathy, (CIDP), are triggered by an autoimmune response against speciic components of the axon or the myelin sheath 19 whereas hereditary motor and sensory neuropathies (HMSN), also known as Charcot-Marie-Tooth (CMT) disease, are caused by mutations in particular axon- or myelin-related genes 20. Acquired neuropathies. GBS is the most common cause of acute autoimmune neuromuscular paralysis, counting approximately 1.5/105 cases worldwide 21. Clinically it is characterized by sensory disturbance, arelexia and muscle weakness often leading to paralysis of legs, arms and respiratory muscles. he acute phase of GBS occurs within 10-14 days after trivial infections and lasts for about 4 weeks. he outcome is highly variable, ranging from good recovery (most cases), to total paralysis (20% of cases) and even death (5-10% in the UK) 22. 15 Introduction Disease Chapter 1 need to re-navigate the paths followed before injury and generate speciic synapses with exactly the same muscle ibers they had previously innervated. In this task they are guided by attractive and repulsive molecular cues 16,17 but physical factors also play a major role 18. It is important to note that excessive axonal branching at the injury site may impair the accuracy of target reinnervation. Since motor and sensory axons have equally little ability to identify Schwann cell tubes leading to their proper target, axons could be redistributed to wrong pathways (misdirection) and a single neuron could send axonal processes to multiple antagonistic muscles (hyperinnervation), impairing functional recovery. hus, both a physiological control of axonal branching and the maintenance of intact endoneurial tubes are of high importance for regeneration of the adult peripheral nerve. Histologically, regeneration is marked by the appearance of regenerative clusters of axons which are branches of the originally injured axon. Initially, these branches reside within a single Schwann cell but they are later separated by radial sorting. Once the 1:1 relationship between Schwann cell and axon is established, the pro-myelinating Schwann cell begins to ensheath the axon and starts to form myelin and the basal lamina tube. At this stage, regenerative clusters appear within adjacent Schwann cells as groups of small caliber, thinly myelinated axons (Figure 3). Continue axonal growth, at a rate of approximately 2.5mm a day, eventually leads to the connection of one branch of the axon to its target. At this point, the rest of the branches are eliminated while the remaining axon thickens. Figure 4. Chromosomal localization of HMSN genes and loci. Gene is annotated on the left and disease loci on the right side. Adapted from: http://www.molgen.ua.ac.be/CMTMutations/Home/IPN.cfm). A number of GBS variants exist. he most common form - acute inlammatory demyelinating polyneuropathy (AIDP) - is pathologically characterized by segmental demyelination of most myelinated axons but spares the extraocular nerves 23. he axonal variant of GBS - acute motor axonal neuropathy (AMAN) - afects the axolemma leading to axonal injury with consequent conduction block which is reversible. In the worst of cases, AMAN leads to complete axonal transection which, depending on the site, can result in poor (proximal injury) or good (distal injury) recovery 24. he regional variants of GBS afect speciic areas of the body with the most common form - Miller Fisher Syndrome (MFS) - afecting the extraocular nerves 25. he chronic counterpart of GBS is chronic inlammatory demyelinating polyradiculoneuropathy (CIDP) characterized by slowly progressive weakness and sensory dysfunction of the limbs. CIDP afects all age groups, including the irst year of life, and in 80% of cases spontaneous recovery is not complete. Many insights into the immunobiology of acquired demyelinating neuropathies come from studies of the experimental autoimmune neuritis (EAN) animal models of human GBS. EAN can be actively induced in rats by immunization with puriied PNS myelin as well as bovine or recombinant human P2-protein 26. Histologically, EAN peripheral nerve and nerve root biopsies are characterized by early iniltration of lymphocytes followed by a massive invasion of macrophages 27. T-lymphocytes play 16 17 Introduction Hereditary neuropathies. Hereditary motor and sensory neuropathies (HMSNs) are a clinically and genetically heterogeneous group of diseases which afect 1:2500 to 1:10000 people in the western countries 39. HMSNs have been originally described on the basis of their clinical features by Charcot and Marie in France 40 and Tooth in England 41 from which they derived their traditional name of Charcot-Marie-Tooth (CMT) disease. Today mutations in more than 30 genes expressed by the Schwann cell or the axon have been identiied Chapter 1 an active role in the pathogenesis of EAN. Adoptive transfer (AT-EAN) of a T cell line speciic for P2-protein is sufficient to induce EAN in rats 28 and T-cell deicient rats do not develop EAN upon active immunization 29. Once activated and diferentiated, T-cells secrete cytokines such as interleukine-2 (IL-2) and tumor necrosis factor-α (TNFα) and mediate the recruitment of macrophages and B-cells into the nerve. Although not the irst cell type to invade the nerve, macrophages represent the predominant cell population in EAN nerves. hey play a role as both antigen presenting cells and phagocytes. Antigen presentation is achieved by the major histocompatibility complex (MHC) class II antigen which is constitutively expressed by resident macrophages. MHC presumably mediates the diferentiation of autoreactive T-cells which secrete cytokines involved in the recruitment of hematogenous macrophages. he active phagocytotic role of macrophages has been demonstrated. Macrophage depletion, with either silica dust or dichlormethylene diphosphonate-containing liposomes, ameliorates the EAN disease phenotype 30,31. In addition to their direct action, macrophages release arachidonic acid metabolites and reactive oxygen species. Selective inhibition of either of those products reduces the severity of EAN 32,33. he discovery of axonal glycolipids-speciic antibodies in EAN animal models and the ability to induce axonal GBS in rabbits sensitized with GM1 gangliosides have considerably advanced our knowledge of the mechanisms responsible for the development of GBS and its regional pattern of clinical involvement 34,35. Notably, speciic anti-ganglioside antibodies correlate highly with discrete variants of GBS: anti-GM1, anti-GD1a, anti-GD1b and anti-GalNAcGD1a antibodies are highly associated with AMAN 36; anti-GQ1a, anti-GD3 and anti-GD1b with MFS 37 whereas anti-GM1 IgG/IgM antibodies are closely linked with AIDP and CIDP 38. Gangliosides are especially enriched in neuronal tissues, primarily localized at raft domains on the synaptic plasma membranes. he carbohydrate moieties of gangliosides are structurally similar to that of microbial glycans such as the lipo-oligosaccharides expressed on the surface of the bacterium Campylobacter jejuni, highly associated with the development of GBS. his mimicry may explain the development of autoimmunity. he immune response, mediated by the anti-ganglioside antibody binding, ights the infection but also attacks peripheral nerves 35. Table 1. Hereditary motor and sensory neuropathies (or CMT) Phenotype CMT1 CMT2 CMT3 CMT4 CMTX Type Form Gene Location CMT1a demyelinating PMP22 17p11 CMT1b demyelinating MPZ 1q22 CMT1c demyelinating LITAF 16p13 CMT1d demyelinating EGR2 10q21 CMT2a1 axonal KIF1Bbeta 1p36 CMT2a2 axonal MFN2 1 CMT2b axonal Rab7 3q13 CMT2c axonal n.d.* 12q23-q24 CMT2d axonal GARS 7p15 CMT2e axonal NEFL 8p21 CMT2f axonal HSPB1/HSP27 7q11 CMT2g axonal n.d.* 12q12 DSS MPZ, PMP22, PRX, EGR2, CX32, GDAP1 CMT4a demyelinating GDAP1 8q13 CMT4b1 demyelinating MTMR2 11q22 CMT4b2 demyelinating MTMR13/SBF2 11p15 CMT4c demyelinating SH3TC2 5q23 CMT4d (HMNSLom) demyelinating NDRG1 8q24 CMT4e demyelinating EGR2 10q21 CMT4f demyelinating PRX 19q13 CMT4h demyelinating FGD4 12q12 CMT4j demyelinating FIG4 6q21 demyelinating GJB1/Cx32 HNPP PMP22 Xq13 17p11 *n.d., no genes are yet identiied for this type of CMT as cause of the disease (http://www.molgen.ua.ac.be/CMTMutations/ and igure 4). Intriguingly, mutations in one gene can give rise to multiple diseases and multiple genes may be involved in a single disorder (reviewed in 42). Exemplar is the peripheral myelin protein (PMP) 22. Genetic alterations of the PMP22 gene can lead to three diferent diseases: a duplication of a 1.5Mb region of chromosome 17p11 gives rise to HMSN1a 43; a deletion of the same region causes hereditary neuropathy with liability to pressure palsies (HNPP) 44; point mutations in the PMP22 gene give rise to the more severe Dejerine- 18 19 Introduction Figure 5. Light microscopy of cross-sections of human nerve biopsy, showing a normal morphology of axons and myelin in the healthy nerve whereas loss of myelin and axon, onion bulbs and high amount of collagen are evident in the neuropathy (HMSN 1a) patient. Chapter 1 Sottas syndrome (DSS) phenotype with onset of neuropathy in the early neonatal period 45. he classiication of HMSNs is currently based on mutations in deined Schwann cell or neuronal genes determining the demyelinating or axonal forms of the disease (Table 1). he primary demyelinating forms of neuropathies are classiied as CMT1. hese include CMT1a and 1b, 1c and 1d. CMT1a, caused by a PMP22 gene duplication, is the most common form of demyelinating neuropathy. CMT1b is caused by mutations in the myelin protein zero (MPZ, P0). CMT1c is linked to lipopolysaccharide-induced TNF factor (LITAF) mutations, whereas early growth response (EGR) 2, a transcription factor for Schwann cell development, results in CMT1d. Clinical disease hallmark include slowly and progressive muscle weakness and atrophy, and sensory impairment. Most patients have skeletal deformities such as pes cavus resulting from an early onset and longstanding mismatch between extensor and lexor forces on the foot muscles. Pathologically CMT1 is characterized by severe loss of myelin and myelinated axons. In CMT1 a pathological hallmark are the so called “onion bulbs”, half moon-shaped Schwann cell processes resulting from excessive Schwann cell proliferation 42 (Figure 5). he primary axonal forms of neuropathies fall within the CMT2 phenotype and are caused by mutations in many diferent genes. Mitofusin (MFN) 2 mutations are the most common cause of CMT2. Clinically, axonal neuropathies are characterized by length-dependent weakness and sensory loss, reduced evoked compound muscle action potential but normal nerve conduction velocities 42. he histology shows prominent axonal swelling, loss of large caliber myelinated ibers and regenerative cluster of axons 46. he CMT3 form of neuropathy includes the Dejerine-Sottas Syndrome (DSS). he group of CMT4 comprises of recessive demyelinating HMSNs. Motor nerve conduction velocities are severely reduced 47. Also in this type of CMT, many diferent genes with diferent functions can be involved. In some cases like HMSN Lom (CMT4D) there are additional disease manifestations. In most patients, deafness develops within the third decade of life. he X-linked form of CMT, CMTX, is linked to mutations in the gene encoding for the gap junction protein connexin 32 (CX32, GJB1) 42. he observation that the same gene mutation can give rise to a variety of disease phenotypes, even within the same family, indicates the involvement of additional factors in determining the severity of the disease. Although inlammation is not considered a criterion to classify HMSN, inlammatory foci have been described in nerve biopsies from HMSN patients and a few patients have responded to antiinlammatory or immunomodulatory treatment that ameliorated the symptoms 48-52. hese observations make the immune system an interesting candidate as modiier of inherited peripheral neuropathies. Another argument for the role of the immune system in HMSNs comes from studies in mice models of the disease (reviewed in 53). he peripheral nerve of mice heterozygously deicient for P0 (P0+/-) or lacking the gap junction protein connexin 32 (Cx32-/-) or carrying four copies of the human PMP22 gene (C61), models for HMSN1B, HMSN1X and HMSN1A respectively, showed elevated number of CD8 lymphocytes and macrophages 54-56. Interestingly, a quantitative analysis of immune cells in P0+/mice of diferent ages showed that the number of macrophages is high at age 4 months - typical disease onset - whereas the number of T-lymphocytes increases starting at age 6 months, indicating that the early increase in the number of macrophages does not result from T-cells iniltration. his is in contrast to the classical EAN model of immune-mediated neuropathies, in which T-lymphocytes are the irst cell types to invade the nerve 57. he endoneurial macrophage population of P0+/- mice mainly consists of resident macrophages which proliferate and become activated within the nerve 58. he factors responsible for such increase and activation are not known. Monocyte chemoattractant protein-1 (MCP-1), released by Schwann cells, has been proposed as a possible candidate 59. hese immune cells seem to play a pathogenic role, at least in certain mouse models of HMSNs. When either P0+/- or Cx32-/- mice were cross-bred with an immunodeicient strain of mice lacking the recombination activation gene (RAG) 1 (Rag1-/-), the peripheral nerve of the double mutants showed no lymphocytes, reduced number 20 he complement system is part of the innate immune response. Originally described as “complement” to humoral immunity, today it represents a central efector mechanism of the innate immune system, defending the host against infections, bridging innate and adaptive immunity and disposing of immune complexes and apoptotic cells 62,63. To date complement counts more than 30 soluble and membrane bound proteins which mediate activation and regulation of the proteolytic cascade to inely balance the elimination of invading pathogens and the protection of the host by limiting complement deposition on healthy tissue. However, if this delicate balance is disrupted, the complement system may cause injury and contribute to the pathogenesis of various diseases. Origins of complement research Complement research began in the 1890s. Von Fodor 64, Nuttall 65 and Buchner 66 all observed that fresh, not heated, serum lyses bacteria. Buchner named the heatsensitive bactericidal serum factor “alexin” (Greek, “to ward of”), for protective substance. In the following years, Bordet 67 showed that the bacteriolytic activity of the serum required the complementary action of the heat-labile component and the heat-stable antibody. he term “alexin” was replaced with the term “complement” by Ehrlich 68. hese discoveries were rewarded with Nobel prizes to Paul Ehrlich (1854-1915) in 1907 and Jules Bordet (1870-1961) in 1919. he inding that complement consisted of more than just one factor began when Buchner 66 showed that serum diluted with water separated into an insoluble, “euglobulin”, and soluble fraction, “pseudoglobulin”. In the laboratory of Ehrich, Ferrata 69 proved that both fractions were necessary for complement activity. Contemporarily 21 Introduction The complement system Chapter 1 of macrophages and a signiicant amelioration of the demyelinating phenotype as documented by morphometry and electrophysiological recordings 60. When P0+/- mice were cross-bred with a mouse line lacking the macrophage colony-stimulating factor (M-CSF), critical for macrophage growth and diferentiation, the P0+/-/M-CSF-/- double mutants showed a decrease in the number of macrophage which resulted in the alleviation of the pathological abnormalities, proving an active role of macrophages in determining the demyelinating phenotype 61. Although the immune system has been implicated in certain type of hereditary neuropathies, a possible involvement of the innate immune system in HMSN has not yet been shown and it is one of the subjects of this thesis. Brand 70 demonstrated that euglobulin, which he termed “midpiece”, reacted irst with the antibody whereas the pseudoglobulin, which he termed “endpiece”, reacted second to produce lysis. his was the discovery of C1 and C2. he discovery of the third complement component began with the observation that cobra venom destroyed complement activity 71. A few years later, Omorokow 72 showed that cobra venum removed a heat stable factor from the serum. Apparently, this same component was removed by absorption by yeast cells 73. his factor was further characterized and named C3 by Whitehead et al. 74. In 1926, the same research group in Scotland described the fourth component 75. hey believed that serum lipids were involved in the complement-mediated lysis of bacteria so they tested the efects of ammonia extraction of serum on complement activity. Ammonia destroyed a heat stable protein which however did not reconstitute the activity removed by yeast. his was the discovery of C4. he following 30 years of complement research were primarily based on functional measurements performed with “R” reagents, serum fraction of partially inactivated serum to selectively study the activity of single components. hese studies resulted in the determination of the order of reactivity of the complement protein: C1, C4, C2 and C3. he identiication of C3 as plasma protein by Müller-Eberhard et al 76 changed complement research. he novel approach was based on the puriication and characterization of the other complement components as proteins, their biological activity and their interactions. By the mid-1960s, the complement cascade counted 11 components whose sequence of reaction was also known: C1q, C1r, C1s, C4, C2, C3, C5, C6, C7, C8 and C9 77. Suspicion of multiple ways of activating complement dates back to the observations of Ritz 78 and Coca 73 but the deinite demonstration of an “alternative” way to the “classical” way of activating complement came from the Gewurz group 79 who showed that bacterial lipopolysaccharides consumes C3-9 without consuming C1 or C4 and by the work of Frank et al 80 who showed that C4 deicient guinea pig serum retained the ability to activate complement. he search for proteins involved in the alternative pathway was very competitive: Boenisch and Alper 81 demonstrated the existence of a new protein which they termed glycine-rich beta glycol-protein (GBG); Götze and Müller-Eberhard 82 named it C3 proactivator (C3PA) but it was Goodkofsky and Lepow 83 who called it with the name which is now used, Factor B. he component upstream of Factor B was initially isolated by Alper and Rosen 84 who called it glycine-rich beta glycoproteinase (GB-Gase) and later described by Müller-Eberhard and Götze 85 who named it C3PA convertase. his was Factor D. he mechanism of alternative pathway activation became clear with the discovery of Factor I, initially called Konglutinogen activating factor (KAF). It was described as an enzyme acting upon C3b, whose absence determined agglutination of cells treated 22 Activation of the complement system is rapid and efficient. Soluble complement components are present in the blood, body luids and tissues to readily trigger a defense reaction against external (i.e. pathogens) or internal (i.e. autoimmunity) danger signals. he complement system can be activated via three routes: the classical, the lectin and the alternative pathway (Figure 6). he classical pathway is activated by the recognition of an antigen-antibody complex by C1q. Upon binding, C1r cleaves C1s which in turn cleaves C2 and C4 into a small (C2b, C4a) and a large fragment (C2a, C4b). C2a and C4b together form the C3 convertase. he lectin pathway is triggered by binding of mannose-binding lectins MBLs to certain carbohydrates expressed on the pathogen surface. his activates the MBL-associated serine protease (MASP) 2, cleaving C4 and C2 91. he alternative pathway starts by spontaneous low-rate hydrolysis of C3 generating C3(H2O) which binds to factor B, permitting cleavage by factor D to form the luid-phase C3 convertase C3(H2O)Bb. his enzyme cleaves C3 and deposits C3b on surfaces where, in the absence of C inhibitors such as factor H, it binds and catalyses cleavage of factor B to form surface bound C3 convertase C3bBb 92. Irrespectively of the pathway involved, activation of the complement system leads to the cleavage of C3 and C5, generating the potent chemo-attractants C3a and C5a as well as the C5b fragment. his initiates the assembly of the C5b-9 membrane attack complex (MAC) which is a lipophilic complex inserted into the pathogens cell membrane, leading to cell lysis 92. Recently, additional venues of complement activation have been proposed. he so called “C2 bypass” pathway consists of the direct cleavage of C3 by MASP-2 of the lectin pathway, bypassing formation of the C3 convertase 93. Direct cleavage of C3 and C5 by noncomplement proteins such as lysosomal enzymes released from neutrophils, kallikrein, part of the kinin and ibrinolysis systems, or trombin has also been shown 23 Introduction A tight balance of activation and regulation Chapter 1 with bovine serum as a source of complement, a process known as conglutination 86. he observation that heated serum depleted of Factor I would cleave all C3 and Factor B, suggested that the activation of the alternative pathway did not require initiation but it was spontaneously active and continuously regulated by an inhibitory pathway. Inactivation of the inhibitory pathway would allow a positive feedback ampliication loop for C3 cleavage 87. Visualization by electron microscopy of the pores characteristic of complement lysis represented a major advance in the ield of complement research 88. It was later shown that the presence of C567 did not lyse cells until the last two components were added to the serum 89. More than 10 years later, Podack and Tschopp 90 showed that the pores are circular polymers of C9. his was the membrane attack complex (MAC). Figure 6. Factors and pathways of the complement system. and the route named “extrinsic protease” pathway 94. Lastly, properdin stabilizes the C3 convertase on the pathogen surface but it also seems to act as a pattern recognition molecule and directly induces C3 convertase on foreign surfaces 95,96 (Figure 6). A sophisticated regulatory mechanism allows the complement system to rapidly attack invading pathogens while protecting host cells from its detrimental efects. his is achieved via the coordination of time, location and molecular interactions. After activation, several thousand C3b molecules deposit every minute on a cell. In theory, this opsonization is not speciically directed against foreign cells. However, the thioster bond of C3b, which enables it to covalently bind to hydroxyl groups on nearby carbohydrates and protein-acceptor groups, has a short half life, limiting its action to the site of activation. If the acceptor molecule is on the host cell, a set of soluble and membrane-bound negative regulators inactivate and degrade the activated complement components. On the other hand, positive regulators such as properdin, stabilize the convertase on foreign cells 97. hese regulatory complement components either induce an accelerated decay of the convertase or act as cofactor for Factor I. Decay accelerating factor (DAF/CD55) and C4-binding protein (C4BP) accelerate decay of the convertase; membrane cofactor protein (MCP/CD46) acts together with Factor I to degrade C3b to its inactive form iC3b; complement receptor 1 (CR1/CD35) and Factor H can do both. In addition, both membrane-bound and soluble CD59 (sCD59), prevent the formation of the MAC by inserting between the C8 and C9 subunit 97 (Figure 7). 24 Chapter 1 Introduction Figure 7. Regulators of the complement system. Complement in inflammation Activated complement components regulate various stages of an inlammatory reaction. hese events are mediated by the potent complement anaphylatoxins C3a and C5a which propagate the immune reaction by binding to their receptors (C3aR, C5aR, C5L2) on the host cell. Macrophages, widely distributed in connective tissues and various organs (i.e. lung, liver, spleen), express complement receptors. he interactions between complement receptors on macrophages and opsonins on the target cell mediate activation of the phagocyte and secretion of cytokines and chemokines (reviewed by 98). Anaphylatoxins activate also mast cells to release histamine, tumor necrosis factor-α (TNF-α), cytokines and chemokines, mediating vasodilation and leukocyte migration from the blood stream to the site of inlammation 99. Complement is also involved in the disposal of immune complexes, necrotic and apoptotic cells which are usually generated during an inlammatory reaction 62,63. he clearance of immune complexes is facilitated by maintaining their solubility through the binding of the C1 complex, C4 and C3 to the antigen. his prevents an increase in the size of the opsonized complex which is easily recognized by phagocytes and readily removed to limit inlammation and prevent the propagation of injury on neighboring tissues. he removal of necrotic and apoptotic cells is critical for the termination of inlammation and prevention of autoimmunity. Dying cells undergo several changes to signal their removal to phagocytic cells. hese changes involve modiications of the 25 plasma membrane resulting in the exposure of self-antigens, normally sequestered within the viable cell, and the internalization of proteins normally expressed on the cell surface. he presentation of “eat me” signals and the down-regulation of “don’t eat me” signals trigger the binding of a number of opsonins to mediate the removal of dying cells. his is a key process in the maintenance of tissue homeostasis and normally does not require complement binding. However, during overwhelming apoptosis or impaired phagocytosis complement initiation factors can bind dying cells to ensure proper disposal of self-antigens 100, avoiding the generation of an autoimmune reaction against the host. Complement in inflammatory and neurodegenerative disorders Disruption of the delicate balance between complement activation and regulation is implicated in the pathogenesis, propagation and exacerbation of numerous diseases afecting a variety of organs (Figure 8). Paradoxically both, overactivation or deiciency in complement components may contribute to tissue injury. Excessive complement activation results from the propagation of an inlammatory reaction or from alterations in the expression and function of complement regulatory proteins. Complement activation, especially C5a production, seems to play a major role in the pathogenesis of inlammatory disorders including ischemia/reperfusion injury, sepsis, acute lung injury, allergy and asthma 101. It is also involved in a number of neurodegenerative disorders, including Alzheimer’s, Huntington’s, Parkinson’s, Creutzfelt-Jakob disease and amyotrophic lateral sclerosis (ALS) (reviewed in 102-104) and neuroinlammatory diseases such as multiple sclerosis (MS) 105. In his seminal observations, Alzheimer 106 described a close association between amyloid plaques and brain phagocytes. Evidence accumulated over the past two decades suggested that inlammation may contribute to AD pathogenesis and implicates complement as a potential mediator of the inlammatory response 107,108. Complement transcripts and proteins are upregulated in AD brains, in some cases up to 80-fold 109. Deposits of complement components including C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 and C9 have been found in pyramidal neurons 110 and plaques 111. Activated complement fragments have been described in close association with tangles and plaques, and MAC deposits have been found on dystrophic neurites 112 whereas the complement regulatory protein CD59 is strongly decreased in the prefrontal cortex and hippocampus of AD patients 113. Both, the classical and alternative pathways of complement have been implicated in AD 114,115 and seem to mediate neuronal injury via MAC-induced neurite disintegration 116 and increase in the level of reactive oxygen species 117. he role of complement in AD remains however controversial since evidence mainly from AD animal models treated with complement inhibitors have shown an increase in amyloid β accumulation and neuronal degeneration suggesting 26 Figure 8. Summary of diseases in which the complement system has been implicated. Whether the complement system is involved in peripheral neuropathies is one of the aims of this thesis. Chapter 1 27 Introduction the possibility of a neuroprotective role of complement in AD pathology, probably by reducing accumulation and promoting the clearance of amyloid and degenerating neurons 118. Deposits of complement components and activated microglia have been reported in the substantia nigra of patients with sporadic and familial Parkinson’s disease 119. he striatum, neurons, myelin and astrocytes of Huntington’s disease patients and the extracellular deposits of the prion protein in Creutzfelt-Jakob disease are marked by deposits of activated complement fragments 120,121. Gene expression array analysis of motor neurons from mice with a mutation in the superoxidedismutase 1 (SOD1) gene, involved in a familial form of ALS, identiied the induction of components of the classical pathway of complement 122,123 but the question whether the complement system plays a detrimental or neuroprotective role still remains open. Complement activation also occurs in the brain of MS patients 124,125 and a pathological role has been shown in the experimental autoimmune encephalomyelitis (EAE) model of MS 126,127. Dysfunction of regulatory proteins may lead to complement-mediated tissue injury. Mutations and polymorphisms in the genes encoding factor H, factor I or MCP are associated with atypical hemolytic-uremic syndrome (aHUS), a rare disorder which leads to acute renal failure 128. Age-related macula degeneration (AMD), the major cause of blindness in Western countries, has been associates with a tyrosine-histidine polymorphism at amino acid 402 of factor H 129. Mutations in the C1 inhibitor gene, which either result in low C1 inhibitor levels or abolish its activity as a serine protease, are responsible for hereditary angioedema (HAE) 130. Deiciency of CD59 has been reported as a cause for the intravascular hemolysis characteristic in paroxysmal nocturnal hemoglobinuria (PNH) 131. Finally, deiciency of complement components appears to be a major contributor to the development of autoimmune diseases. Complement deiciency may impair the clearance of immune complexes and the removal of apoptotic and necrotic cells. his generates an autoimmune reaction against self-antigens which are exposed by the dying cells. In addition, complement lowers the threshold for B and T cells activation. In the absence of complement, an augmentation of the inlammatory response may thus erroneously target host tissues. In summary, a tight balance between complement activation and regulation is essential to protect and maintain tissue homeostasis. Aim and outline of this thesis he aim of this thesis is to understand the role of the complement system in the degeneration, regeneration and disease processes of the peripheral nervous system. We used the crush injury model of Wallerian degeneration (WD) to exemplify axonal degeneration and regeneration and we focused on the hereditary forms of demyelinating neuropathies as disease status of the peripheral nerve. Chapter 2 reviews the current knowledge on the expression of complement components in the peripheral nerve, their putative physiological role, implication in injury and disease and therapeutic approaches aimed at regulating excessive complement activation. Chapter 3 dissects the function of the upstream complement components from the cytolitic efect of the terminal membrane attack complex (MAC) during WD. In Chapter 4 a “reverse proof of principle” study describes the efects of lack of complement regulation during WD. 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Br.J.Haematol. 137, 181-192. 35 Introduction Mead R.J., Singhrao S.K., Neal J.W., Lassmann H., & Morgan B.P. (2002) he membrane attack complex of complement causes severe demyelination associated with acute axonal injury. J.Immunol. 168, 458-465. Chapter 1 127.