The C3a and C5a anaphylatoxins signal through the C3aR and C5aR1 G-protein coupled receptors, but we lack a clear understanding of their function and downstream effector mechanisms in the CNS.
Certainly both anaphylatoxins can promote the production of cytokines and inflammatory mediators, and can direct and activate leukocytes, but they can also affect neuronal survival and synaptic plasticity. In addition, expression of C3aR on both neurons and glial cells suggests that C3a may play a role beyond immunological protection Several studies have investigated common and different neuroprotective mechanisms of C3a and C5a.
C3a was found to be involved in basal and ischemia-induced neurogenesis that was inhibited in C3-deficient mice and in mice treated with C3a receptor antagonist C3aRA In this latter study, C5a was found to inhibit DNA fragmentation and pro-caspase-3 activation in neuronal and hippocampal cultures. Both C3a and C5a were found to increase the gene expression of neuronal growth factor in microglia, an effect mediated by C3aR and C5aR1 and inhibited by pertussis toxin that blocks G-protein Gi coupled receptors 35 , Both C3a and C5a contributed to neuroprotection from glutamate-induced excitotoxicity, although each peptide had a different specific protective pattern.
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C3a was only protective against N -methyl- d -aspartate NMDA in the presence of astrocytes, and did not contribute to neuroprotection against Kainaite-induced excitotoxicity Also, intraventricular infusion of both Kainic acid and C5a in mice reduced caspase-3 activation and neuronal apoptosis 38 , and C5aR1-deficient mice were shown to have increased susceptibility to Kainaite-induced excitotoxicity compared to wild type; treatment of wild-type mice with C5a reversed the glutamate-induced decrease in GluR2 receptor and reduced neuronal apoptosis More recently, C5a was shown to reduce extracellular glutamate accumulation through the upregulation of glutamate transporter GLT-1 in microglia, suggesting a distinct mechanism of protection against excitotoxicity The interplay between complement system and other immune components in normal and pathological brain.
The complement system has long been recognized as a potential therapeutic target for the reduction of secondary damage and improvement of outcome after stroke. Research has focused on identifying the role of complement in brain ischemia-reperfusion injury IRI and investigating how complement inhibition affects outcome in models of stroke. There follows a summary of studies that have been performed in vitro , in animal models of ischemic and hemorrhagic stroke, and in human stroke.
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Oxygen-glucose deprivation of cultured neuronal cells is a widely used in vitro model for cerebral ischemia, a procedure that results in both apoptotic and necrotic cell death. Upon hypoxic insult, neuronal cultures have been shown to overexpress several complement proteins. Both mRNA and protein levels of C1q were elevated in rat neuronal cells exposed to hypoxia, and newly produced C1q preferentially deposited on hypoxic neurons, serving as both a primary opsonin and an activator of the complement cascade Similarly, mouse and rat neuronal cell cultures showed increased C3 expression in response to hypoxia, a response that was shown to be associated with activation of caspase-3, a marker for apoptosis.
Both C3 expression and caspase-3 activation were reduced with intravenous immunoglobulin IVIG treatment, suggesting that IVIG may represent an interventional therapy for stroke 42 , In addition, blocking C5a signaling by the use of C5aR1 antagonist or the use of neurons from C5aR1-deficient mice reduced ischemia-induced apoptosis in murine neuronal cultures indicating a pathogenic role for C5a 44 , The neuroprotective effect of C5aR1 antagonism could be enhanced with hypothermia without alteration in C5aR1 levels, suggesting a putative therapeutic advantage of coupling both treatments On the other hand, human neurons were found to express the complement inhibitors CD59, CD46 membrane cofactor protein and CD55 decay accelerating factor , and hypoxic insult neither altered inhibitor expression nor the deposition of C3d, suggesting that human neurons are protected from the effects of C3 opsonization and the MAC Animal models of ischemic stroke involve transient or permanent occlusion of the middle cerebral artery or common carotid artery, or cerebral clot embolization.
Notably, the advantage of the cerebral embolization model, although more difficult and less commonly utilized, is that it better allows the evaluation of the effect of potential adjuvant therapies to tissue plasminogen activator t-PA , the only approved treatment for acute stroke.
As a plasma protease, t-PA is capable of proteolytically activating components of the complement system via the recently recognized extrinsic pathway. In support of this, an early study reported that after cerebral embolization rabbits treated with t-PA had higher levels of C3 and C5 compared to vehicle Interestingly, complement depletion in the same model using cobra venom factor CVF did not have any effect on infarct size in the presence or absence of t-PA treatment The use of CVF in rodent models of transient ischemia consistently demonstrates a protective effect of complement depletion.
Rats subjected to bilateral transient common carotid artery occlusion and pretreated with CVF had a better outcome compared to control treated rats in terms of somatosensory evoked potentials CVF also reduced infarct volume and neuronal atrophy after rat transient middle cerebral artery occlusion MCAO , as well as after neonatal rat hypoxia 50 , However, in permanent ischemia rat model, CVF did not effectively reduce infract volume The prominent role of reperfusion in the activation of plasma complement proteins near ischemic tissue may explain why complement depletion did not alter outcomes in models utilizing permanent ischemia.
CR2-Crry, targeted inhibitor of complement activation. Following stroke, damage to the blood—brain barrier will allow access of hematogenous complement proteins to the CNS, but there is evidence that increased local expression of complement proteins contribute to secondary injury after ischemia.
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For example, expression of C1q, C3, and CD11b a component of complement receptor 3 expressed on phagocytes is upregulated in response to ischemia in models of transient MCAO 54 , 58 , 60 , Interestingly, the increase in C1q mRNA expression was specific to microglia and did not occur in neurons or astrocytes 54 , but C1q protein in the ischemic brain co-localized with the neuronal marker MAP2, suggesting that C1q is produced by microglia in response to ischemia and accumulates on neuronal cells However, despite increased expression of C1q, the role of the classical pathway in post-ischemic injury is not completely clear since whereas C1q deficiency protects against murine neonatal hypoxic-ischemic brain injury 79 , 85 , it does not protect against murine transient MCAO In neurodegenerative diseases, C1q and or C3 opsonization of synapses stimulates microglial phagocytosis, and complement-mediated synaptic clearance precedes neuronal loss 21 , During development, however, C1q and C3 opsonins have been shown to play an essential role in synaptic pruning and neuron remodeling A feature of cerebral IRI is that it carries characteristics of both neurodegenerative disease and developmental mechanisms.
Activation of inflammatory cascades after stroke causes significant secondary injury resulting in loss of both synapses and neurons in the penumbra area. Following this early insult, stroke is characterized by a window of neuroplastic response that mimics early developmental mechanisms including axonal growth and sprouting, and formation of new synapses and intra-cortical projections 87 — 89 [see reviews 90 , 91 ].
An important, yet unexplored question, is whether inhibition of the complement system after cerebral ischemia and reperfusion reduces loss of synaptic connections in the ischemic penumbra or whether this inhibition will prevent adequate synaptic pruning during rehabilitation induced cortical re-organization. In an influential paper on the application of complement inhibition in stroke, Huang et al. Soluble CR1 inhibits all complement pathways at the C3 activation step, and the sLex carbohydrate moiety binds to both P and E selectin, adhesion molecules that are upregulated on activated endothelium.
Following MCAO and reperfusion, sCR1 modestly reduced ischemic injury while sCR1-sLex markedly diminished infarct volume, improved neurological outcome, and inhibited leukocyte migration Thus, decoration sCR1 with the sLex moiety increased the efficacy of complement inhibition by targeting the inhibitor to selectin-expressing activated brain endothelial surfaces. A more recent study demonstrated that a truncated form of sCR1 also significantly improved outcome in a rat model of transient MCAO Nevertheless, studies in a non-human primate stroke model failed to reproduce the earlier findings of sCR1 and sCR1-sLex treatment that was reported in mice Similarly, the therapeutic value of sCR1-sLex failed to translate to baboons in a study by Ducruet et al.
In this latter study, sCR1-sLex reduced complement hemolytic activity, but failed to improve neurological outcome; in fact, treatment increased infarct volume in baboons. One possible explanation for this outcome is that sCR1-sLex has multivalent selectin-targeting sites and may promote homotypic platelet—platelet aggregation, and potentially thrombosis.
Another strategy used to target complement inhibition to an anatomical site is to link a complement inhibitor to a recombinant portion of complement receptor type 2 CR2. Ligands for CR2 are C3 activation products that become covalently attached at sites of complement activation Acute outcomes after a single injection of CR2-Crry were comparable to those in C3-deficient mice It has also been shown that targeted complement inhibition with CR2-Crry does not impair host susceptibility to infection, unlike systemic inhibition of C3 deficiency, a potentially important consideration for stroke patients who are at increased risk of infection The sCR1 and Crry inhibitors described above are pan complement inhibitors.
Interventional studies in experimental models of stroke have also been performed with C1-inhibitor C1-inh that inhibits only the classical and lectin pathways. Taken together with the data reported above that C1q deficiency is not protective in murine models of ischemic stroke, these data suggest a role for the lectin pathway in promoting cerebral IRI. Of note, however, all of the studies with C1-inh evaluated acute outcome after stroke, and it is possible that C1q-mediated uptake of apoptotic cells, an important anti-inflammatory and reparatory mechanism, may play a role in outcome in the subacute phase after stroke.
Further investigation has indeed demonstrated an important role for the lectin pathway in murine models of stroke. MBL deficiency resulted in significantly reduced infarct volumes, neurological impairment, and C3 deposition, whereas reconstitution of MBL-deficient mice with MBL resulted in larger infarct volumes Thus, lectin pathway inhibition appears to be a therapeutic target with a wide therapeutic window.
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It should be noted, nevertheless, that Morrison et al. With regard to the different subacute outcomes in lectin pathway deficient vs. Unlike deficiency, acute temporary lectin pathway inhibition would have minimal impact on the lectin pathway in the subacute phase. The alternative complement pathway can be spontaneously activated, but it also serves as an amplification loop for the classical or lectin pathways.
This is likely the reason that although the classical and lectin pathways can variously play essential roles in autoimmune, inflammatory, and ischemic disease models, the alternative pathway is nearly always required for full in vivo expression of injury 93 , Together these data indicate that the alternative pathway is not alone sufficient to initiate complement activation, but that it acts to propagate cerebral injury via amplification of the cascade. In unpublished work, we have shown that unlike CR2-fH, CR2-Crry does not provide sustained protection into the subacute phase after stroke.
This may reflect an advantage of not blocking all complement pathways in order to maintain some level of complement activation that may contribute to homeostatic or repair processes. With regard to the terminal complement pathway, data indicate that the MAC is an important mediator of IRI in several organs and tissues, including spinal cord injury 95 and traumatic brain injury In agreement with this conclusion, mice deficient in CD59, a membrane-bound inhibitor of the MAC, do not display exacerbated cerebral injury in the same model Studies on the anaphylatoxins, C3a and C5a, have indicated important roles for these peptides in cerebral IRI.
In a transient MCAO model, C3-deficient mice and wild-type mice treated with a C3a receptor antagonist C3aRA showed significant improvements in infarct volume and neurological outcomes, with less granulocyte infiltration.
Furthermore, the effect was reversed when C3-deficient mice were reconstituted with C3 The different effects of C3aRA in the two models may be related to inhibition of C3a-dependent activation of blood-derived immune cells following reperfusion. C5aR1-deficient mice are also protected against cerebral IRI following transient MCAO 44 , but pretreatment of wild-type mice with C5aRA did not consistently improve neurological outcome or infarct volume 42 , Interestingly, C5aRA was also found to improve outcome in animal models of hemorrhagic stroke, either alone or in combination with C3aRA 53 , 67 , These studies on the role of C3a and C5a in stroke pathogenesis were performed by analyzing acute outcomes, and it remains unclear what effect interfering with C3a- and C5a-mediated signaling would have on subacute and chronic outcomes.
A concern with inhibiting C3a and C5a signaling, especially at later time points after stroke, is the effect on neurogenesis and cognitive performance, since both anaphylatoxins have implicated roles in basal and stress-induced neurogenesis 32 — This may indicate that a titrated reduction of C3a-dependent signaling may limit the pro-inflammatory effects of C3a especially in models that include reperfusion , while at the same time maintain the homeostatic effects of C3a on neurogenesis. So what is the complement-activating event after stroke?
Seminal studies by Williams et al. The intestinal target of this mAb was identified as non-muscle myosin 98 , and antibodies to this post-ischemic neoepitope have additionally been shown to play a role in hind-limb and myocardial IRI Although the early studies attributed IgM-mediated activation of the classical pathway as an initiating event in IRI, more recent studies have demonstrated that IRI is driven by IgM-mediated activation of the lectin pathway, at least in the organ systems that have been investigated 99 , , As with intestinal IRI, Rag1-deficient mice are protected from cerebral IRI, and two IgM monoclonal antibodies were identified that reconstituted cerebral IRI in Rag1-deficient mice; these monoclonal antibodies recognized annexin IV and a subset of phospholipids, and thus identified danger-associated molecular determinants expressed after stroke in mice Post-ischemic blockade of annexin IV or phospholipid neoepitopes thus represents a potential therapeutic strategy for inhibiting complement activation and reducing cerebral IRI.
The same two post-ischemic neoepitopes are also expressed in the intestine and heart [ 57 , Circulation, in press] after ischemia and reperfusion, indicating that similar pathophysiologically important epitopes recognized by IgM natural antibodies are expressed in different organs and tissues. Furthermore, it has been shown that human endothelial cells HUVEC express these two neoepitopes after exposure to hypoxia Triggers of complement activation after cerebral ischemia-reperfusion injury. Ischemic insult induces expression of neoepitopes or danger-associated molecular patterns DAMPs on the surface of stressed endothelial cells.
The exposed DAMPs are recognized by circulating natural self-reactive antibodies, principally IgM, which triggers complement activation. Although IgM binds C1q, it appears to be the binding of MBL and activation of the lectin pathway that drives ischemia and reperfusion injury in the organs systems examined, including the brain. Complement can be also activated through direct binding of C1q to apoptotic cells, as well as through C-reactive protein-induced complement activation. There are no reported clinical trials on the use of complement inhibition in stroke, a direct consequence of the aforementioned conflicting pre-clinical data.
Before an intervention can enter a clinical trial, stringent criteria recommended by the stroke therapy academic industry roundtable STAIR must be met. In brief, there should be consistent pre-clinical data in different animal species and in animal cohorts that represent the human stroke predisposed population. Such populations include both sexes, adult and elderly age groups, and patients with comorbidities such as hypertension and diabetes Thus, studies on the role of complement in human stroke are correlational.
Studies on gene polymorphisms at complement gene loci, associated polymorphisms at the C5 and factor H gene loci with increased incidence of cerebrovascular accidents , Among 16 single nucleotide polymorphisms SNPs at the C3 gene locus, two SNPs rs and rs were associated with ischemic stroke MBL genotype is also associated with stroke severity. In two cohorts of stroke patients, MBL-sufficient phenotypes were associated with larger infarct and less favorable outcome compared to MBL-deficient phenotypes 77 , Prospective studies on men with advanced atherosclerosis, higher levels of C4 and C5 are associated with the incidence of stroke Similarly, a prospective study of healthy men showed that C4 and C3 levels correlated with other cardiovascular risk factors, and that very high C4 levels may predict the incidence of stroke C4 was also shown to be a stroke predictor in men with known or suspected coronary artery disease Compared to healthy volunteers, stroke patients were found to have higher plasma levels of C3a, C3, C4, C5, factor B, and the terminal complement complex — Serum levels of C3, C3c, and C4 were also associated with increased stroke severity in cardio-embolic stroke patients , Despite these human studies that report alterations in serum complement levels in stroke patients and the association with outcome, the studies remain correlative and cannot demonstrate a definitive role for complement in the pathogenesis of stroke.
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The predominant focus of studies on complement and stroke has been on the role of complement in perpetuating the inflammatory cascade and contributing to neuronal death. By comparison, very few studies have investigated the putative protective role that complement may play in ischemic rescue and ischemia-induced neurogenesis. The great majority of reports investigate acute-phase outcomes where complement blockade prevents activation of pro-inflammatory mechanisms. However, as indicated by a few studies, the same favorable outcomes may not occur in the subacute and chronic phases after stroke, when complement opsonins may be important for mediating anti-inflammatory events such as removal of debris, and when anaphylatoxins may contribute to homeostatic pro-survival and neuro-regenerative effects.
It will be important to investigate how manipulation of the complement system impacts subacute and chronic outcomes, as well as to evaluate areas away from the ischemic core. Investigation of chronic outcomes may reveal a role for complement in maintaining neuronal survival after acute damage has been resolved, and possible mechanisms may include stimulation of neuronal growth factor production by astrocytes and microglia 35 , 36 , or reduction of glutamate-induced excitotoxicity 37 — Stroke and brain injury are associated with a window of synaptic plasticity after injury that contributes to neurological recovery 88 , While various mechanisms have been implicated in this process, a role for complement has not been investigated.
Based on the essential role of complement in synaptic pruning during CNS development, complement may also play a role in synaptic pruning and hippocampal plasticity after stroke, and may thus contribute to improved neurological outcome and facilitate rehabilitation. Going forward, the design of experimental models to assess longer endpoints and multiple outcome measures after stroke may reveal a more diverse role for complement in ischemic injury and rescue.
Future studies should also investigate the role of different complement components in early and delayed pathophysiological events. One area of investigation that has received little attention is the role of complement opsonins in early neuronal and synaptic loss after stroke, as well as the role of these opsonins in the resolution of inflammation and synaptic re-wiring that can occur later.
We also have an incomplete understanding of the role of the anaphylatoxins in stroke.
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They are implicated in recruiting and activating immune cells acutely, as well as stimulating neuronal genesis and migration in delayed phases after stroke. It is also not clear whether C3a and C5a have redundant, overlapping, or possibly divergent effects. Tools to address these questions are available in the form of genetically deficient mice and inhibitors that block the generation of specific complement activation products or that antagonize complement receptors, and it will be important for future studies to better evaluate long-term effects and outcomes after stroke.
In addition, postmortem studies have identified both complement and IgM deposition in the human brain after stroke This suggests that complement activation and deposition occurs in the human brain similarly to that in experimental models. Thus, given the correlation between complement activity and stroke outcome in human patients, the use of complement inhibition holds promise as a potential therapeutic intervention.
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