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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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Related in: MedlinePlus

Cell associated C1q complement staining. Paraffin wax sections A1, A2 and A3 (case MS160_S3, active case). A1 shows immunolabelling with anti-C1q in plaque (P) and peri-plaque (PP) areas. A2 shows C1q immunopositive debris (brown) within foamy macrophages localized in the centre of an active plaque. A3 shows C1q immunolabelled on myelin. B1 paraffin wax section double immunolabelled with anti-C1q (grey) and anti-HLA-DR (brown) confirms C1q immunopositive debris located within HLA positive foamy macrophages cells; (case MS160_S3, active plaque). B2 and B3 also show paraffin wax sections with immune double staining of anti-C1q and anti-HLA-DR (case MS230_s2, chronic active plaque). B2 shows HLA-DR positive cells (brown) closely associated with C1q positive myelin (grey) within the white matter. B3 shows co-localisation of C1q on HLA-DR positive microglia (arrows) in the peri-plaque. C1 (case MS372_22, chronic active plaque) and C2 (case MS230_S2, chronic active plaque, stained using immunofluorescence (IFC)) show double labelling for C1q and GFAP, with colocalisation of C1q (grey) and GFAP (brown) in some but not all cells (insert in C1 and arrow in C2 highlighting colocalisation). IFC in figure D1 (case MS377_S2, active plaque) and D2 (case MS160_S1, active plaque) demonstrate anti-HLA-DR positive macrophages closely associated with C1q positive myelin (arrow). Inset in D2 shows C1q immunopositive debris within an HLA-DR immunolabelled macrophage (arrow). E1 shows a myelin sheath at edge of an active plaque staining with anti-MOG (grey) and C1q (brown) (case MS160_S3, arrow); in the same area E2 (IFC, case MS160_S3) shows a myelin sheath with positive anti-C1q immunolabelling. Figures F1 (MS160_S3/1; chronic active plaque) and F2 (MS336_1; active plaque), captured with confocal laser scanning microscopy, show disrupted myelin, immuno-positive for C1q associated with anti-SMI-32 immunopositive non-phosphorylated axon profiles in chronic and active areas. Scale bars are shown for each plate.
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Fig4: Cell associated C1q complement staining. Paraffin wax sections A1, A2 and A3 (case MS160_S3, active case). A1 shows immunolabelling with anti-C1q in plaque (P) and peri-plaque (PP) areas. A2 shows C1q immunopositive debris (brown) within foamy macrophages localized in the centre of an active plaque. A3 shows C1q immunolabelled on myelin. B1 paraffin wax section double immunolabelled with anti-C1q (grey) and anti-HLA-DR (brown) confirms C1q immunopositive debris located within HLA positive foamy macrophages cells; (case MS160_S3, active plaque). B2 and B3 also show paraffin wax sections with immune double staining of anti-C1q and anti-HLA-DR (case MS230_s2, chronic active plaque). B2 shows HLA-DR positive cells (brown) closely associated with C1q positive myelin (grey) within the white matter. B3 shows co-localisation of C1q on HLA-DR positive microglia (arrows) in the peri-plaque. C1 (case MS372_22, chronic active plaque) and C2 (case MS230_S2, chronic active plaque, stained using immunofluorescence (IFC)) show double labelling for C1q and GFAP, with colocalisation of C1q (grey) and GFAP (brown) in some but not all cells (insert in C1 and arrow in C2 highlighting colocalisation). IFC in figure D1 (case MS377_S2, active plaque) and D2 (case MS160_S1, active plaque) demonstrate anti-HLA-DR positive macrophages closely associated with C1q positive myelin (arrow). Inset in D2 shows C1q immunopositive debris within an HLA-DR immunolabelled macrophage (arrow). E1 shows a myelin sheath at edge of an active plaque staining with anti-MOG (grey) and C1q (brown) (case MS160_S3, arrow); in the same area E2 (IFC, case MS160_S3) shows a myelin sheath with positive anti-C1q immunolabelling. Figures F1 (MS160_S3/1; chronic active plaque) and F2 (MS336_1; active plaque), captured with confocal laser scanning microscopy, show disrupted myelin, immuno-positive for C1q associated with anti-SMI-32 immunopositive non-phosphorylated axon profiles in chronic and active areas. Scale bars are shown for each plate.

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 C1q complement staining. Paraffin wax sections A1, A2 and A3 (case MS160_S3, active case). A1 shows immunolabelling with anti-C1q in plaque (P) and peri-plaque (PP) areas. A2 shows C1q immunopositive debris (brown) within foamy macrophages localized in the centre of an active plaque. A3 shows C1q immunolabelled on myelin. B1 paraffin wax section double immunolabelled with anti-C1q (grey) and anti-HLA-DR (brown) confirms C1q immunopositive debris located within HLA positive foamy macrophages cells; (case MS160_S3, active plaque). B2 and B3 also show paraffin wax sections with immune double staining of anti-C1q and anti-HLA-DR (case MS230_s2, chronic active plaque). B2 shows HLA-DR positive cells (brown) closely associated with C1q positive myelin (grey) within the white matter. B3 shows co-localisation of C1q on HLA-DR positive microglia (arrows) in the peri-plaque. C1 (case MS372_22, chronic active plaque) and C2 (case MS230_S2, chronic active plaque, stained using immunofluorescence (IFC)) show double labelling for C1q and GFAP, with colocalisation of C1q (grey) and GFAP (brown) in some but not all cells (insert in C1 and arrow in C2 highlighting colocalisation). IFC in figure D1 (case MS377_S2, active plaque) and D2 (case MS160_S1, active plaque) demonstrate anti-HLA-DR positive macrophages closely associated with C1q positive myelin (arrow). Inset in D2 shows C1q immunopositive debris within an HLA-DR immunolabelled macrophage (arrow). E1 shows a myelin sheath at edge of an active plaque staining with anti-MOG (grey) and C1q (brown) (case MS160_S3, arrow); in the same area E2 (IFC, case MS160_S3) shows a myelin sheath with positive anti-C1q immunolabelling. Figures F1 (MS160_S3/1; chronic active plaque) and F2 (MS336_1; active plaque), captured with confocal laser scanning microscopy, show disrupted myelin, immuno-positive for C1q associated with anti-SMI-32 immunopositive non-phosphorylated axon profiles in chronic and active areas. Scale bars are shown for each plate.
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Related In: Results  -  Collection

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Show All Figures
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Fig4: Cell associated C1q complement staining. Paraffin wax sections A1, A2 and A3 (case MS160_S3, active case). A1 shows immunolabelling with anti-C1q in plaque (P) and peri-plaque (PP) areas. A2 shows C1q immunopositive debris (brown) within foamy macrophages localized in the centre of an active plaque. A3 shows C1q immunolabelled on myelin. B1 paraffin wax section double immunolabelled with anti-C1q (grey) and anti-HLA-DR (brown) confirms C1q immunopositive debris located within HLA positive foamy macrophages cells; (case MS160_S3, active plaque). B2 and B3 also show paraffin wax sections with immune double staining of anti-C1q and anti-HLA-DR (case MS230_s2, chronic active plaque). B2 shows HLA-DR positive cells (brown) closely associated with C1q positive myelin (grey) within the white matter. B3 shows co-localisation of C1q on HLA-DR positive microglia (arrows) in the peri-plaque. C1 (case MS372_22, chronic active plaque) and C2 (case MS230_S2, chronic active plaque, stained using immunofluorescence (IFC)) show double labelling for C1q and GFAP, with colocalisation of C1q (grey) and GFAP (brown) in some but not all cells (insert in C1 and arrow in C2 highlighting colocalisation). IFC in figure D1 (case MS377_S2, active plaque) and D2 (case MS160_S1, active plaque) demonstrate anti-HLA-DR positive macrophages closely associated with C1q positive myelin (arrow). Inset in D2 shows C1q immunopositive debris within an HLA-DR immunolabelled macrophage (arrow). E1 shows a myelin sheath at edge of an active plaque staining with anti-MOG (grey) and C1q (brown) (case MS160_S3, arrow); in the same area E2 (IFC, case MS160_S3) shows a myelin sheath with positive anti-C1q immunolabelling. Figures F1 (MS160_S3/1; chronic active plaque) and F2 (MS336_1; active plaque), captured with confocal laser scanning microscopy, show disrupted myelin, immuno-positive for C1q associated with anti-SMI-32 immunopositive non-phosphorylated axon profiles in chronic and active areas. Scale bars are shown for each plate.
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