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NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG.

Walker-Caulfield ME, Guo Y, Johnson RK, McCarthy CB, Fitz-Gibbon PD, Lucchinetti CF, Howe CL - J Neuroinflammation (2015)

Bottom Line: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO).This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905, USA.

ABSTRACT

Background: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO.

Methods: Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining.

Results: Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.

Conclusions: We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

No MeSH data available.


Related in: MedlinePlus

Granulocytic infiltrate occurs in the absence of demyelination, terminal complement complex formation, and overt tissue destruction in NMO white matter. a H&E staining reveals robust perivascular inflammation in the white matter of autopsy tissue collected from an NMO patient (scale bar = 100 μM). b An enlarged view of (a) and the magnified inset confirm the presence of eosinophils as a component of this perivascular infiltration (scale bar = 50 μM). c Staining for the astrocyte marker GFAP demonstrates the presence of reactive astrocytes with abnormal morphology in close association with granulocytic perivascular infiltration in a section consecutive to a. The inset highlights the increased size of a GFAP+ astrocyte (scale bar = 100 μM). d The perivascular granulocytic infiltration and astrocyte reactivity are present in a non-demyelinating NMO lesion as shown by the presence of intact proteolipid protein (PLP) staining in a section consecutive to a and c (scale bar = 100 μM). e A second example of perivascular granulocytic infiltration involving neutrophils and eosinophils (inset) in the white matter of an NMO patient is shown by H&E staining. The inset highlights the presence of eosinophils (scale bar = 50 μM). f Staining for the astrocyte marker GFAP in a section consecutive to e confirms the presence of numerous reactive astrocytes proximal to the perivascular inflammation (scale bar = 50 μM). g PLP staining reveals that in a section consecutive to (e) and (f) the myelin is intact, indicating that granulocytic infiltrate is found in the absence of demyelination (scale bar = 50 μM). h The complete absence of staining for the terminal complement protein C9neo in a section consecutive to e, f, and g shows that granulocytic recruitment to this site is not dependent on formation of the terminal complement complex (scale bar = 100 μM)
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Fig1: Granulocytic infiltrate occurs in the absence of demyelination, terminal complement complex formation, and overt tissue destruction in NMO white matter. a H&E staining reveals robust perivascular inflammation in the white matter of autopsy tissue collected from an NMO patient (scale bar = 100 μM). b An enlarged view of (a) and the magnified inset confirm the presence of eosinophils as a component of this perivascular infiltration (scale bar = 50 μM). c Staining for the astrocyte marker GFAP demonstrates the presence of reactive astrocytes with abnormal morphology in close association with granulocytic perivascular infiltration in a section consecutive to a. The inset highlights the increased size of a GFAP+ astrocyte (scale bar = 100 μM). d The perivascular granulocytic infiltration and astrocyte reactivity are present in a non-demyelinating NMO lesion as shown by the presence of intact proteolipid protein (PLP) staining in a section consecutive to a and c (scale bar = 100 μM). e A second example of perivascular granulocytic infiltration involving neutrophils and eosinophils (inset) in the white matter of an NMO patient is shown by H&E staining. The inset highlights the presence of eosinophils (scale bar = 50 μM). f Staining for the astrocyte marker GFAP in a section consecutive to e confirms the presence of numerous reactive astrocytes proximal to the perivascular inflammation (scale bar = 50 μM). g PLP staining reveals that in a section consecutive to (e) and (f) the myelin is intact, indicating that granulocytic infiltrate is found in the absence of demyelination (scale bar = 50 μM). h The complete absence of staining for the terminal complement protein C9neo in a section consecutive to e, f, and g shows that granulocytic recruitment to this site is not dependent on formation of the terminal complement complex (scale bar = 100 μM)

Mentions: To determine whether granulocytic infiltration in NMO is dependent on formation of the terminal complement complex, demyelination, or tissue destruction, as the conventional model of NMO pathogenesis suggests, we semi-quantitatively assessed the extent of eosinophil and neutrophil infiltration in 1048 regions in 337 blocks from 23 NMO patients. In a subset of lesions, we observed granulocyte infiltration (Fig. 1a, b, e) in the absence of terminal complement complex formation, demyelinating activity, and tissue destruction (Fig. 1). C9neo served as a marker for terminal complement deposition, and although granulocyte infiltration was statistically associated with the presence of complement deposition (p = 0.012), 31 % of regions with mild infiltration, 20 % of regions with moderate infiltration, and 4 % of regions with marked infiltration were negative for C9neo (Fig. 1h). With regard to demyelinating activity, we found a statistically significant association between active demyelination and granulocytic infiltration (p < 0.001). However, the presence of granulocytes was not entirely dependent on demyelinating activity, as we observed mild, moderate, or marked infiltration in 9 % of NMO lesions devoid of demyelination (Fig. 1d, g). Finally, in regions with mild, moderate, or marked granulocyte infiltration, there was no statistically significant association between the extent of tissue damage and infiltration (p = 0.99) (Fig. 1), suggesting that granulocytic trafficking is not strictly in response to the presence or extent of tissue damage in NMO. These observations support the hypothesis that recruitment of neutrophils and eosinophils into the CNS may be one of the earliest consequences of NMO IgG binding to the surface of astrocytes. Furthermore, we observed profound astrocyte reactivity in proximity to infiltrating neutrophils and eosinophils (Fig. 1c, f), supporting our hypothesis that early astrocytic responses to NMO IgG may drive granulocytic recruitment and infiltration that precedes irreversible astrocytic death, demyelination, and tissue damage.Fig. 1


NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG.

Walker-Caulfield ME, Guo Y, Johnson RK, McCarthy CB, Fitz-Gibbon PD, Lucchinetti CF, Howe CL - J Neuroinflammation (2015)

Granulocytic infiltrate occurs in the absence of demyelination, terminal complement complex formation, and overt tissue destruction in NMO white matter. a H&E staining reveals robust perivascular inflammation in the white matter of autopsy tissue collected from an NMO patient (scale bar = 100 μM). b An enlarged view of (a) and the magnified inset confirm the presence of eosinophils as a component of this perivascular infiltration (scale bar = 50 μM). c Staining for the astrocyte marker GFAP demonstrates the presence of reactive astrocytes with abnormal morphology in close association with granulocytic perivascular infiltration in a section consecutive to a. The inset highlights the increased size of a GFAP+ astrocyte (scale bar = 100 μM). d The perivascular granulocytic infiltration and astrocyte reactivity are present in a non-demyelinating NMO lesion as shown by the presence of intact proteolipid protein (PLP) staining in a section consecutive to a and c (scale bar = 100 μM). e A second example of perivascular granulocytic infiltration involving neutrophils and eosinophils (inset) in the white matter of an NMO patient is shown by H&E staining. The inset highlights the presence of eosinophils (scale bar = 50 μM). f Staining for the astrocyte marker GFAP in a section consecutive to e confirms the presence of numerous reactive astrocytes proximal to the perivascular inflammation (scale bar = 50 μM). g PLP staining reveals that in a section consecutive to (e) and (f) the myelin is intact, indicating that granulocytic infiltrate is found in the absence of demyelination (scale bar = 50 μM). h The complete absence of staining for the terminal complement protein C9neo in a section consecutive to e, f, and g shows that granulocytic recruitment to this site is not dependent on formation of the terminal complement complex (scale bar = 100 μM)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Granulocytic infiltrate occurs in the absence of demyelination, terminal complement complex formation, and overt tissue destruction in NMO white matter. a H&E staining reveals robust perivascular inflammation in the white matter of autopsy tissue collected from an NMO patient (scale bar = 100 μM). b An enlarged view of (a) and the magnified inset confirm the presence of eosinophils as a component of this perivascular infiltration (scale bar = 50 μM). c Staining for the astrocyte marker GFAP demonstrates the presence of reactive astrocytes with abnormal morphology in close association with granulocytic perivascular infiltration in a section consecutive to a. The inset highlights the increased size of a GFAP+ astrocyte (scale bar = 100 μM). d The perivascular granulocytic infiltration and astrocyte reactivity are present in a non-demyelinating NMO lesion as shown by the presence of intact proteolipid protein (PLP) staining in a section consecutive to a and c (scale bar = 100 μM). e A second example of perivascular granulocytic infiltration involving neutrophils and eosinophils (inset) in the white matter of an NMO patient is shown by H&E staining. The inset highlights the presence of eosinophils (scale bar = 50 μM). f Staining for the astrocyte marker GFAP in a section consecutive to e confirms the presence of numerous reactive astrocytes proximal to the perivascular inflammation (scale bar = 50 μM). g PLP staining reveals that in a section consecutive to (e) and (f) the myelin is intact, indicating that granulocytic infiltrate is found in the absence of demyelination (scale bar = 50 μM). h The complete absence of staining for the terminal complement protein C9neo in a section consecutive to e, f, and g shows that granulocytic recruitment to this site is not dependent on formation of the terminal complement complex (scale bar = 100 μM)
Mentions: To determine whether granulocytic infiltration in NMO is dependent on formation of the terminal complement complex, demyelination, or tissue destruction, as the conventional model of NMO pathogenesis suggests, we semi-quantitatively assessed the extent of eosinophil and neutrophil infiltration in 1048 regions in 337 blocks from 23 NMO patients. In a subset of lesions, we observed granulocyte infiltration (Fig. 1a, b, e) in the absence of terminal complement complex formation, demyelinating activity, and tissue destruction (Fig. 1). C9neo served as a marker for terminal complement deposition, and although granulocyte infiltration was statistically associated with the presence of complement deposition (p = 0.012), 31 % of regions with mild infiltration, 20 % of regions with moderate infiltration, and 4 % of regions with marked infiltration were negative for C9neo (Fig. 1h). With regard to demyelinating activity, we found a statistically significant association between active demyelination and granulocytic infiltration (p < 0.001). However, the presence of granulocytes was not entirely dependent on demyelinating activity, as we observed mild, moderate, or marked infiltration in 9 % of NMO lesions devoid of demyelination (Fig. 1d, g). Finally, in regions with mild, moderate, or marked granulocyte infiltration, there was no statistically significant association between the extent of tissue damage and infiltration (p = 0.99) (Fig. 1), suggesting that granulocytic trafficking is not strictly in response to the presence or extent of tissue damage in NMO. These observations support the hypothesis that recruitment of neutrophils and eosinophils into the CNS may be one of the earliest consequences of NMO IgG binding to the surface of astrocytes. Furthermore, we observed profound astrocyte reactivity in proximity to infiltrating neutrophils and eosinophils (Fig. 1c, f), supporting our hypothesis that early astrocytic responses to NMO IgG may drive granulocytic recruitment and infiltration that precedes irreversible astrocytic death, demyelination, and tissue damage.Fig. 1

Bottom Line: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO).This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905, USA.

ABSTRACT

Background: Astrocytes expressing the aquaporin-4 water channel are a primary target of pathogenic, disease-specific immunoglobulins (IgG) found in patients with neuromyelitis optica (NMO). Immunopathological analyses of active NMO lesions highlight a unique inflammatory phenotype marked by infiltration of granulocytes. Previous studies characterized this granulocytic infiltrate as a response to vasculocentric complement activation and localized tissue destruction. In contrast, we observe that granulocytic infiltration in NMO lesions occurs independently of complement-mediated tissue destruction or active demyelination. These immunopathological findings led to the hypothesis that NMO IgG stimulates astrocyte signaling that is responsible for granulocytic recruitment in NMO.

Methods: Histopathology was performed on archival formalin-fixed paraffin-embedded autopsy-derived CNS tissue from 23 patients clinically and pathologically diagnosed with NMO or NMO spectrum disorder. Primary murine astroglial cultures were stimulated with IgG isolated from NMO patients or control IgG from healthy donors. Transcriptional responses were assessed by microarray, and translational responses were measured by ELISA. Signaling through the NFκB pathway was measured by western blotting and immunostaining.

Results: Stimulation of primary murine astroglial cultures with NMO IgG elicited a reactive and inflammatory transcriptional response that involved signaling through the canonical NFκB pathway. This signaling resulted in the release of pro-granulocytic chemokines and was inhibited by the clinically relevant proteasome inhibitors bortezomib and PR-957.

Conclusions: We propose that the astrocytic NFκB-dependent inflammatory response to stimulation by NMO IgG represents one of the earliest events in NMO pathogenesis, providing a target for therapeutic intervention upstream of irreversible cell death and tissue damage.

No MeSH data available.


Related in: MedlinePlus