There is on-going debate surrounding the contribution of inflammation to the relentless axonal degeneration seen in MS patients, clinically most apparent in progressive disease; absence of immune cells and lack of efficacy of anti-inflammatories has led some to postulate a complex and as yet undefined neurodegenerative process to explain the relentless brain atrophy . We show that complement proteins, activation products, regulators and receptors are markers of CNS innate immune activation, present in active plaques but persisting even in late stage disease when plaques become inactive, indicating that synthesis, activation and regulation of complement are all on-going in plaque and peri-plaque areas of brain and spinal cord in late-stage MS.
In chronic active and inactive plaques, complement markers occurred in the absence of other inflammation markers, including lymphocytes, plasma cells and foamy macrophages; demonstrating that progression of inflammation in MS CNS does not rely on infiltrating cells; once initiated, inflammation can be driven by innate immune mechanisms such as complement. These findings support our previous studies showing systemic complement activation and regulation in MS throughout the disease course ; in particular, increased serum fH levels were specific to progressive disease and predicted relapse , suggesting an active role in late stage disease.
The profile of complement immunolabelled cells was similar in patients with active and chronic active plaques but markedly different in patients with other neuroinflammatory diseases - encephalitis, Alzheimer’s disease and ischaemic infarction. In MS, complement staining was predominantly associated with GFAP + astrocytes in plaque and peri-plaque areas; in contrast, in neurological controls, cellular complement staining associated with microglia in WM and neurones in GM. In encephalitis and ischaemic infarction cases, the time from pathological insult to death was short in comparison to Alzheimer’s disease and MS where complement expression and activation is maintained over years. In ischaemic infarction there is catastrophic blood brain barrier (BBB) disruption, facilitating entry of complement; in MS, Alzheimer’s disease and encephalitis where the degree of BBB disruption is uncertain, local synthesis may be an important contributor and it is known that glial cells can produce all complement components needed for terminal pathway activation .
Evidence implicating the classical pathway in MS pathogenesis was described over thirty years ago. Immune complexes were demonstrated in MS CSF and brain with associated binding of C1q and classical pathway activation ; in parallel, it was shown that oligoclonal immunoglobulins were present in CSF in most MS patients , the result of intrathecal synthesis . Even in the absence of antibody, the classical pathway can be triggered by C1q directly binding damaged myelin . Here we confirm that C1q is a powerful disease marker, present in all MS cases and minimal in controls. C1q immunolocalized to reactive astrocytes, microglia and myelin in peri-plaque and plaque areas; immunolabelling does not distinguish between protein on and in cells - it is entirely possible that astrocyte staining for C1q and other complement proteins represents biosynthesis, especially in inactive chronic lesions where BBB breakdown is minimal. In contrast, astrocyte- and myelin-associated C3- and C4-derived activation fragments and TCC in MS brain is strong evidence of ongoing classical pathway activation and implies that at least some of the C1q is on the astrocyte surface and activating. The alternative pathway marker Bb was positive in the majority of MS cases, suggesting that amplification through this pathway was a significant contributor.
Complement regulators assessed in this study showed increased immunolabelling in plaque and peri-plaque areas indicating the ongoing capacity for local complement inhibition throughout MS disease course. In light of our published finding that the relative plasma levels of the variants of regulator fH alter in progressive MS , the Tyr402His polymorphic variants (minor allele frequency 0.3) were separately measured; remarkably, the relative staining of Tyr and His variants was markedly different between MS cases and controls, suggesting that there were qualitative differences in fH variant expression and/or deposition between cases and controls with more frequent cell-associated fH, and in particular the His402 variant, in MS. The functional consequences and relevance to disease progression of this remain to be ascertained; however, in age-related macular degeneration (AMD), differential binding of the fH-Tyr402His variants in retina has been demonstrated and proposed as the mechanism by which fH-His402 is risk for AMD . Of note, we previously demonstrated increased plasma levels of fH and altered Tyr402His ratio in progressive MS with over-representation of His402 in heterozygous patients .
Our data demonstrate considerable heterogeneity of complement immunolabelling in MS cases. Pathological heterogeneity and heterogeneity of complement staining in MS lesions was described by Lucchinetti and colleagues; they proposed four distinct patterns of MS lesions with only 50% of patients showing complement immunolabelling, limited in their study to TCC immunolabelling using the mAb B7, also used in our work [10, 27]. Others have contested this, using a wider range of complement markers and reporting consistent complement immunolabelling in all lesions and cases examined . Our data agree in part with the findings of Lucchineti in that 71% of sections were TCC+; however, all were C1q +, most C3b/iC3b + and all TCC + sections were strongly C3b/iC3b+. Together these data indicate that complement staining is a ubiquitous feature of MS lesions but the pattern of staining is variable. We suggest that TCC is a reliable marker for complement activation in MS; its near-complete absence in controls supports its use in isolation as a marker. In our hands, very few lesions were negative for TCC and C3b/iC3b and these were C1q+, which may also be a useful marker despite its presence in control tissues.
The predominant staining of reactive astrocytes for complement activation products including iC3b and TCC has not previously been described in MS and was disease-specific; complement activation marker staining in neurological controls was extracellular and on microglia/macrophages in the tissue. The data suggest that this astrocyte-restricted pattern of complement staining is specific to demyelinating disease. Complement-mediated astrocytic impairment was recently described in neuromyelitis optica ; our findings imply that this may also be a feature of progressive MS. Complement-stained astrocytes were often co-localised with clusters of activated microglia, a finding that has previously been reported in MS and linked to complement priming of microglia contributing to CNS inflammation . The notion of astrocytes contributing to pathological process in MS by enhancement of immune response and disruption of myelin has recently been raised in a number of studies [30, 31].
Immunolabelling of disrupted myelin in MS lesions, previously cited as evidence of complement-mediated pathology, was limited and variable in this study. Breij et al.  described myelin staining for C3d but not TCC in active lesions. Myelin staining for complement proteins has been suggested to be a non-specific feature ; however, using double immunolabelling we found complement markers co-localized in areas of degenerating myelin and damaged axons in MS sections (Figures 4 and 5), implicating complement in axonal and myelin injury in and around active and chronic plaques. Absence of TCC may reflect efficient regulation of terminal pathway activation at these sites or an inability of TCC/MAC to intercalate into myelin. Clusters of activated microglia were frequently found in close apposition to damaged myelin, likely representing iC3b/CR3-mediated opsonic clearance.
C3aR and C5aR were expressed on microglia in and around the plaque, a finding previously reported in acute plaques [32, 33], but not in chronic active and inactive slowly expanding lesions studied here; indeed, numbers of C3aR/C5aR-positive microglia were higher in chronic/inactive lesions than in the few active plaques observed. The abundance of C3aR/C5aR-positive microglia in the peri-plaque suggests a capacity for locally generated C3a and C5a to recruit and activate microglia even in late lesions.