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Complement activation in multiple sclerosis plaques: an immunohistochemical analysis.

Ingram G, Loveless S, Howell OW, Hakobyan S, Dancey B, Harris CL, Robertson NP, Neal JW, Morgan BP - Acta Neuropathol Commun (2014)

Bottom Line: Inflammation and complement activation are firmly implicated in the pathology of multiple sclerosis; however, the extent and nature of their involvement in specific pathological processes such as axonal damage, myelin loss and disease progression remains uncertain.This study aims to bring clarity to these questions.We describe a detailed immunohistochemical study to localise a strategically selected set of complement proteins, activation products and regulators in brain and spinal cord tissue of 17 patients with progressive multiple sclerosis and 16 control donors, including 9 with central nervous system disease.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK. morganbp@cardiff.ac.uk.

ABSTRACT

Introduction: Inflammation and complement activation are firmly implicated in the pathology of multiple sclerosis; however, the extent and nature of their involvement in specific pathological processes such as axonal damage, myelin loss and disease progression remains uncertain. This study aims to bring clarity to these questions.

Results: We describe a detailed immunohistochemical study to localise a strategically selected set of complement proteins, activation products and regulators in brain and spinal cord tissue of 17 patients with progressive multiple sclerosis and 16 control donors, including 9 with central nervous system disease. Active, chronic active and chronic inactive multiple sclerosis plaques (35 in total) and non-plaque areas were examined.Multiple sclerosis plaques were consistently positive for complement proteins (C3, factor B, C1q), activation products (C3b, iC3b, C4d, terminal complement complex) and regulators (factor H, C1-inhibitor, clusterin), suggesting continuing local complement synthesis, activation and regulation despite the absence of other evidence of ongoing inflammation. Complement staining was most apparent in plaque and peri-plaque but also present in normal appearing white matter and cortical areas to a greater extent than in control tissue. C1q staining was present in all plaques suggesting a dominant role for the classical pathway. Cellular staining for complement components was largely restricted to reactive astrocytes, often adjacent to clusters of microglia in close apposition to complement opsonised myelin and damaged axons.

Conclusions: The findings demonstrate the ubiquity of complement involvement in multiple sclerosis, suggest a pathogenic role for complement contributing to cell, axon and myelin damage and make the case for targeting complement for multiple sclerosis monitoring and therapy.

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Cell associated C3b complement staining. Paraffin wax section of active peri-plaque regions (A, case MS377_S2; B, case MS383_21) immunolabelled with anti-C3b showing cells with astrocytic morphology. Myelin degradation debris (arrows) within macrophages colabelled with anti-C3b (brown) and MOG (grey) are shown within the centre of an active plaque (C, MS160_S3). Immunofluorescent staining: D1 (case MS 225_S13, chronic active plaque) and D2 (case 377_S2, active plaque) showing colabelling of C3b (red) and GFAP + astrocytes (green) in the peri-plaque white matter. E1 (case MS225_S13, chronic active plaque; plaque (P) and peri-plaque (PP)), E2 and E3 (both case MS377_S2, active plaque) show zones of HLA-DR macrophages/microglia and an adjacent area of predominantly astrocytic and axonal C3b immunolabelled structures. In figure E3, note the close approximation of C3b positive axon (arrow) and HLA positive microglial cell. F shows an anti-C3b positive myelin strand (brown, arrow) co-localised with MOG (grey) on an axon bundle within striatum (case MS160_S3, active plaque). Immunofluorescent staining (G), showing anti-C3b immunopositive myelin (red) co-localised with an anti-SMI-32 (green), immunopositive non-phosphorylated axon at the edge of a chronic active white matter lesion (case MS160_S3, active plaque). Scale bars are shown for each slide.
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Fig5: Cell associated C3b complement staining. Paraffin wax section of active peri-plaque regions (A, case MS377_S2; B, case MS383_21) immunolabelled with anti-C3b showing cells with astrocytic morphology. Myelin degradation debris (arrows) within macrophages colabelled with anti-C3b (brown) and MOG (grey) are shown within the centre of an active plaque (C, MS160_S3). Immunofluorescent staining: D1 (case MS 225_S13, chronic active plaque) and D2 (case 377_S2, active plaque) showing colabelling of C3b (red) and GFAP + astrocytes (green) in the peri-plaque white matter. E1 (case MS225_S13, chronic active plaque; plaque (P) and peri-plaque (PP)), E2 and E3 (both case MS377_S2, active plaque) show zones of HLA-DR macrophages/microglia and an adjacent area of predominantly astrocytic and axonal C3b immunolabelled structures. In figure E3, note the close approximation of C3b positive axon (arrow) and HLA positive microglial cell. F shows an anti-C3b positive myelin strand (brown, arrow) co-localised with MOG (grey) on an axon bundle within striatum (case MS160_S3, active plaque). Immunofluorescent staining (G), showing anti-C3b immunopositive myelin (red) co-localised with an anti-SMI-32 (green), immunopositive non-phosphorylated axon at the edge of a chronic active white matter lesion (case MS160_S3, active plaque). Scale bars are shown for each slide.

Mentions: Cell complement immunolabelling within the plaque and peri-plaque areas in MS sections was consistently associated with cells morphologically resembling astrocytes for all complement proteins tested except the anaphylatoxin receptors C3aR and C5aR (Figures 4, 5 and Additional file 3: Figure S2). Double immunolabelling confirmed this observation; C1q and C3b co-localized with GFAP + reactive astrocytes within the plaque and peri-plaque (C3b in this manuscript refers to immunolabelling with the C3b/iC3b-specific monoclonal antibody C3/30) (Figures 4 and 5). C1q and C3b stained reactive astrocytes were often in close proximity to clusters of HLA-positive microglia with activated morphology and macrophages containing complement-stained debris, seen especially in active plaque centres (Figures 4 and 5). C1q also co-localized with HLA + activated microglia, contributing to the increased cell-associated C1q immunolabelling in the peri-plaque (Figures 3 and 4); these cells were also positive for anaphylatoxin receptors C3aR and C5aR (Additional file 3: Figure S2). GFAP positive astrocytes within the GM did not stain for complement; indeed, complement immunolabelling of all complement antibodies within the GM in MS and controls was mainly limited to neurones (Additional file 4: Figure S3).Figure 4


Complement activation in multiple sclerosis plaques: an immunohistochemical analysis.

Ingram G, Loveless S, Howell OW, Hakobyan S, Dancey B, Harris CL, Robertson NP, Neal JW, Morgan BP - Acta Neuropathol Commun (2014)

Cell associated C3b complement staining. Paraffin wax section of active peri-plaque regions (A, case MS377_S2; B, case MS383_21) immunolabelled with anti-C3b showing cells with astrocytic morphology. Myelin degradation debris (arrows) within macrophages colabelled with anti-C3b (brown) and MOG (grey) are shown within the centre of an active plaque (C, MS160_S3). Immunofluorescent staining: D1 (case MS 225_S13, chronic active plaque) and D2 (case 377_S2, active plaque) showing colabelling of C3b (red) and GFAP + astrocytes (green) in the peri-plaque white matter. E1 (case MS225_S13, chronic active plaque; plaque (P) and peri-plaque (PP)), E2 and E3 (both case MS377_S2, active plaque) show zones of HLA-DR macrophages/microglia and an adjacent area of predominantly astrocytic and axonal C3b immunolabelled structures. In figure E3, note the close approximation of C3b positive axon (arrow) and HLA positive microglial cell. F shows an anti-C3b positive myelin strand (brown, arrow) co-localised with MOG (grey) on an axon bundle within striatum (case MS160_S3, active plaque). Immunofluorescent staining (G), showing anti-C3b immunopositive myelin (red) co-localised with an anti-SMI-32 (green), immunopositive non-phosphorylated axon at the edge of a chronic active white matter lesion (case MS160_S3, active plaque). Scale bars are shown for each slide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048455&req=5

Fig5: Cell associated C3b complement staining. Paraffin wax section of active peri-plaque regions (A, case MS377_S2; B, case MS383_21) immunolabelled with anti-C3b showing cells with astrocytic morphology. Myelin degradation debris (arrows) within macrophages colabelled with anti-C3b (brown) and MOG (grey) are shown within the centre of an active plaque (C, MS160_S3). Immunofluorescent staining: D1 (case MS 225_S13, chronic active plaque) and D2 (case 377_S2, active plaque) showing colabelling of C3b (red) and GFAP + astrocytes (green) in the peri-plaque white matter. E1 (case MS225_S13, chronic active plaque; plaque (P) and peri-plaque (PP)), E2 and E3 (both case MS377_S2, active plaque) show zones of HLA-DR macrophages/microglia and an adjacent area of predominantly astrocytic and axonal C3b immunolabelled structures. In figure E3, note the close approximation of C3b positive axon (arrow) and HLA positive microglial cell. F shows an anti-C3b positive myelin strand (brown, arrow) co-localised with MOG (grey) on an axon bundle within striatum (case MS160_S3, active plaque). Immunofluorescent staining (G), showing anti-C3b immunopositive myelin (red) co-localised with an anti-SMI-32 (green), immunopositive non-phosphorylated axon at the edge of a chronic active white matter lesion (case MS160_S3, active plaque). Scale bars are shown for each slide.
Mentions: Cell complement immunolabelling within the plaque and peri-plaque areas in MS sections was consistently associated with cells morphologically resembling astrocytes for all complement proteins tested except the anaphylatoxin receptors C3aR and C5aR (Figures 4, 5 and Additional file 3: Figure S2). Double immunolabelling confirmed this observation; C1q and C3b co-localized with GFAP + reactive astrocytes within the plaque and peri-plaque (C3b in this manuscript refers to immunolabelling with the C3b/iC3b-specific monoclonal antibody C3/30) (Figures 4 and 5). C1q and C3b stained reactive astrocytes were often in close proximity to clusters of HLA-positive microglia with activated morphology and macrophages containing complement-stained debris, seen especially in active plaque centres (Figures 4 and 5). C1q also co-localized with HLA + activated microglia, contributing to the increased cell-associated C1q immunolabelling in the peri-plaque (Figures 3 and 4); these cells were also positive for anaphylatoxin receptors C3aR and C5aR (Additional file 3: Figure S2). GFAP positive astrocytes within the GM did not stain for complement; indeed, complement immunolabelling of all complement antibodies within the GM in MS and controls was mainly limited to neurones (Additional file 4: Figure S3).Figure 4

Bottom Line: Inflammation and complement activation are firmly implicated in the pathology of multiple sclerosis; however, the extent and nature of their involvement in specific pathological processes such as axonal damage, myelin loss and disease progression remains uncertain.This study aims to bring clarity to these questions.We describe a detailed immunohistochemical study to localise a strategically selected set of complement proteins, activation products and regulators in brain and spinal cord tissue of 17 patients with progressive multiple sclerosis and 16 control donors, including 9 with central nervous system disease.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK. morganbp@cardiff.ac.uk.

ABSTRACT

Introduction: Inflammation and complement activation are firmly implicated in the pathology of multiple sclerosis; however, the extent and nature of their involvement in specific pathological processes such as axonal damage, myelin loss and disease progression remains uncertain. This study aims to bring clarity to these questions.

Results: We describe a detailed immunohistochemical study to localise a strategically selected set of complement proteins, activation products and regulators in brain and spinal cord tissue of 17 patients with progressive multiple sclerosis and 16 control donors, including 9 with central nervous system disease. Active, chronic active and chronic inactive multiple sclerosis plaques (35 in total) and non-plaque areas were examined.Multiple sclerosis plaques were consistently positive for complement proteins (C3, factor B, C1q), activation products (C3b, iC3b, C4d, terminal complement complex) and regulators (factor H, C1-inhibitor, clusterin), suggesting continuing local complement synthesis, activation and regulation despite the absence of other evidence of ongoing inflammation. Complement staining was most apparent in plaque and peri-plaque but also present in normal appearing white matter and cortical areas to a greater extent than in control tissue. C1q staining was present in all plaques suggesting a dominant role for the classical pathway. Cellular staining for complement components was largely restricted to reactive astrocytes, often adjacent to clusters of microglia in close apposition to complement opsonised myelin and damaged axons.

Conclusions: The findings demonstrate the ubiquity of complement involvement in multiple sclerosis, suggest a pathogenic role for complement contributing to cell, axon and myelin damage and make the case for targeting complement for multiple sclerosis monitoring and therapy.

Show MeSH
Related in: MedlinePlus