Role for Complement in the Development of Seizures following Acute Viral Infection

Complement, part of the innate immune system, acts to remove pathogens and unwanted host material. Complement is known to function in all tissues, including the central nervous system (CNS). In this study, we demonstrated the importance of the complement system within the CNS in the development of behavioral seizures following Theiler''s murine encephalomyelitis virus (TMEV) infection. C57BL/6 mice, deficient in complement component C3, developed significantly fewer behavioral seizures following TMEV infection, whereas mice depleted of complement component C3 in the periphery through treatment with cobra venom factor had a seizure rate comparable to that of control mice. These studies indicate that C3 participates in the induction of acute seizures during viral encephalitis.The complement system, a component of the innate immune system, functions to recognize and eliminate pathogens and unwanted host material (1). Activation of complement can occur by the classical, alternative, lectin, and terminal pathways (1). The classical pathway is activated by antigen-antibody complexes, some viruses, Gram-negative bacteria, or C-reactive protein complexes (4). The alternative pathway is activated by lipopolysaccharides and polysaccharides on the surfaces of viruses, bacteria, fungi, and parasites. The lectin pathway is activated by mannose or N-acetylglucosamine on the surfaces of bacteria and other pathogens (4). The complement system, consisting of >40 proteins, is highly regulated by the expression of complement inhibitors and complement receptors, as the complement system can have deleterious effects when unregulated (1). Two important steps in the complement cascade are the cleavage of the multifunctional complement proteins C3 and C5 into C3a and C3b proteins and C5a and C5b proteins, respectively. The anaphylatoxins C3a and C5a function to recruit leukocytes and induce inflammation. C3b functions in opsonization, the process of coating pathogens or particulate material with opsonin and thus making it more susceptible to phagocytosis. C5b functions to initiate the assembly of the C5b-C9 complex, the membrane attack complex (MAC), leading to pathogen lysis. Proteins of the complement system are found throughout all tissues and bodily fluids. Although primarily produced by hepatocytes in the liver, other cell types constitutively express low levels of complement proteins (1).Complement proteins are constitutively produced by neurons, microglia, astrocytes, and oligodendrocytes in the central nervous system (CNS) (14, 16, 44; reviewed in references 1 and 13). Astrocytes, the predominant glial cell type in the brain, are comparable to hepatocytes in terms of the number of complement components they produce (4). The levels of various complement mRNAs and proteins are markedly increased in the CNS following viral infection; for example, C1q and C3 proteins and mRNAs are increased in the rat brain following infection with Borna disease virus (11), and C1q protein and mRNA are increased in the rhesus macaque brain following infection with simian immunodeficiency virus (10). In both cases, the increase in the production of C1q in the brain was localized to microglia/macrophages (10, 11). Complement activation has also been shown to be involved in the control of other viral infections, such as the spread of West Nile virus to the CNS (24).We have been studying the role of the innate immune system in the development of acute behavioral seizures following CNS infection of C57BL/6 mice with a neurotropic virus (19, 23). Infection with Theiler''s murine encephalomyelitis virus (TMEV) results in acute seizures developing in more than 50% of C57BL/6 mice (both male and female) generally between days 3 and 10 postinfection (p.i.) (23). Two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and concomitant inflammatory changes in the brain (perivascular cuffing comprised of infiltrating mononuclear cells, infiltration of macrophages, and/or activation of microglial cells and gliosis) were implicated as contributors to the development of acute seizures (19). In contrast, the proinflammatory cytokine IL-1, TMEV-specific CD8⁺ T cells, and viral persistence were discounted as playing a role in seizures (19). It was found that both the pattern of days on which the mice were observed to have seizures and the seizure score (Racine scale) for any given day varied from mouse to mouse (23). Typically, day 3 p.i. was the first day on which a few mice were observed to have seizures, day 6 p.i. was the peak of behavioral seizure activity, and the majority of seizures had a seizure score of 3 (forelimb clonus) and above (score of 4, rearing; score of 5, rearing and falling). The seizures were afebrile and appeared limbic in nature (23). The seizure frequency was observed to be one per mouse per 2-h observation period, and the duration of the seizures was typically 1 to 2 min (23, 31). Mice experiencing seizures were impaired in both coordination and motor function (23).The role of complement in the development of seizures has been studied in humans with Rasmussen''s encephalitis (RE) (41). Although several different viruses have been detected in the tissues of humans with RE by PCR and in situ hybridization, these results are still controversial. Instead it is thought that an autoimmune process underlies RE. Notwithstanding the unknown etiology of RE, the activation of the complement cascade is thought to be a critical component of disease pathogenesis. Several activated components of the complement system (C4, C8, and MAC) were shown to be present in discrete patches of neurons in the cortex of three out of five patients with active RE by immunohistochemistry (41). As a means of demonstrating in vivo that MAC deposition on neurons could trigger seizures, the individual components of the MAC (C5b6, C7, C8, and C9) were sequentially infused into the rat hippocampus, and assembly of the MAC triggered both behavioral and electrographic seizures as well as cytotoxicity (42).In our current study, we examined the role that complement may play in the development of behavioral seizures in the TMEV-induced seizure model. Through the use of mice deficient in complement component C3 and through depletion of complement component C3 in the periphery, we were able to demonstrate the importance of the complement system within the CNS in the development of seizures in the TMEV-induced seizure model.