Inhibition of tumour necrosis factor-α by antisense targeting produces immunophenotypical and morphological changes in injury-activated microglia and macrophages

Damien D Pearse, Francisco C. Pereira, Anna Stolyarova, David J. Barakat, Mary B Bunge

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Microglia respond in a stereotypical pattern to a diverse array of pathological states. These changes are coupled to morphological and immunophenotypical alterations and the release of a variety of reactive species, trophic factors and cytokines that modify both microglia and their cellular environment. We examined whether a microglial-produced cytokine, tumour necrosis factor-α (TNF-α), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF-α antisense deoxyoligonucleotides (ASOs). Microglia and macrophages harvested from 3 d post-contused rat spinal cord were large and rounded (86.3 ± 9.6%). They were GSA-IB4-positive (GSA-IB4+) (Griffonia simplicifolia lectin, microglia specific; 94.8 ± 5.1%), strongly OX-42 positive (raised against a type 3 complement/integrin receptor, CD11b; 78.9 ± 9.1%), ED-1 positive (a lysosomal marker shown to correlate well with immune cell activation; 97.2 ± 2.6%) and IIA positive (antibody recognizes major histocompatibility complex II; 57.2 ± 5.6%), indicative of fully activated cells, for up to 48 h after plating. These cells also secreted significant amounts of TNF-α (up to 436 pg/μg total protein, 16 h). Fluoroscein isothiocyanate-labelled TNF-α ASOs (5, 50 and 200 nM) added to the culture medium were taken up very efficiently into the cells (> 90% cells) and significantly reduced TNF-α production by up to 92% (26.5 pg/μg total protein, 16 h, 200 nM TNF-α ASOs). Furthermore, few of the treated cells at this time were round (5.4 ± 2.7%), having become predominantly spindle shaped (74.9 ± 6.3%) or stellate (21.4 ± 2.7%); immunophenotypically, although all of them remained GSA-IB4 positive (91.6 ± 6.2%), many were weakly OX-42 positive and few expressed either ED-1 (12.9 ± 2.5%) or IIA (19.8 ± 7.4%). Thus, the secretion of TNF-α early in spinal cord injury may be involved in autoactivating microglia/macrophages. However, at the peak of microglial activation after injury, the activation state of microglia/macrophages is not stable and this process may still be reversible by blocking TNF-α.

Original languageEnglish
Pages (from-to)3387-3396
Number of pages10
JournalEuropean Journal of Neuroscience
Volume20
Issue number12
DOIs
StatePublished - Dec 1 2004

Fingerprint

Microglia
Tumor Necrosis Factor-alpha
Macrophages
Wounds and Injuries
Spinal Cord Injuries
Macrophage-1 Antigen
Cytokines
Contusions
Major Histocompatibility Complex
Integrins
Culture Media
Spinal Cord
Proteins
Maintenance
Antibodies

Keywords

  • Cytokines
  • Gene therapy
  • Inflammation
  • Macrophage
  • Microglia
  • Rodent

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Inhibition of tumour necrosis factor-α by antisense targeting produces immunophenotypical and morphological changes in injury-activated microglia and macrophages. / Pearse, Damien D; Pereira, Francisco C.; Stolyarova, Anna; Barakat, David J.; Bunge, Mary B.

In: European Journal of Neuroscience, Vol. 20, No. 12, 01.12.2004, p. 3387-3396.

Research output: Contribution to journalArticle

@article{e379881fa3e44c8cacb7063c44631207,
title = "Inhibition of tumour necrosis factor-α by antisense targeting produces immunophenotypical and morphological changes in injury-activated microglia and macrophages",
abstract = "Microglia respond in a stereotypical pattern to a diverse array of pathological states. These changes are coupled to morphological and immunophenotypical alterations and the release of a variety of reactive species, trophic factors and cytokines that modify both microglia and their cellular environment. We examined whether a microglial-produced cytokine, tumour necrosis factor-α (TNF-α), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF-α antisense deoxyoligonucleotides (ASOs). Microglia and macrophages harvested from 3 d post-contused rat spinal cord were large and rounded (86.3 ± 9.6{\%}). They were GSA-IB4-positive (GSA-IB4+) (Griffonia simplicifolia lectin, microglia specific; 94.8 ± 5.1{\%}), strongly OX-42 positive (raised against a type 3 complement/integrin receptor, CD11b; 78.9 ± 9.1{\%}), ED-1 positive (a lysosomal marker shown to correlate well with immune cell activation; 97.2 ± 2.6{\%}) and IIA positive (antibody recognizes major histocompatibility complex II; 57.2 ± 5.6{\%}), indicative of fully activated cells, for up to 48 h after plating. These cells also secreted significant amounts of TNF-α (up to 436 pg/μg total protein, 16 h). Fluoroscein isothiocyanate-labelled TNF-α ASOs (5, 50 and 200 nM) added to the culture medium were taken up very efficiently into the cells (> 90{\%} cells) and significantly reduced TNF-α production by up to 92{\%} (26.5 pg/μg total protein, 16 h, 200 nM TNF-α ASOs). Furthermore, few of the treated cells at this time were round (5.4 ± 2.7{\%}), having become predominantly spindle shaped (74.9 ± 6.3{\%}) or stellate (21.4 ± 2.7{\%}); immunophenotypically, although all of them remained GSA-IB4 positive (91.6 ± 6.2{\%}), many were weakly OX-42 positive and few expressed either ED-1 (12.9 ± 2.5{\%}) or IIA (19.8 ± 7.4{\%}). Thus, the secretion of TNF-α early in spinal cord injury may be involved in autoactivating microglia/macrophages. However, at the peak of microglial activation after injury, the activation state of microglia/macrophages is not stable and this process may still be reversible by blocking TNF-α.",
keywords = "Cytokines, Gene therapy, Inflammation, Macrophage, Microglia, Rodent",
author = "Pearse, {Damien D} and Pereira, {Francisco C.} and Anna Stolyarova and Barakat, {David J.} and Bunge, {Mary B}",
year = "2004",
month = "12",
day = "1",
doi = "10.1111/j.1460-9568.2004.03799.x",
language = "English",
volume = "20",
pages = "3387--3396",
journal = "European Journal of Neuroscience",
issn = "0953-816X",
publisher = "Wiley-Blackwell",
number = "12",

}

TY - JOUR

T1 - Inhibition of tumour necrosis factor-α by antisense targeting produces immunophenotypical and morphological changes in injury-activated microglia and macrophages

AU - Pearse, Damien D

AU - Pereira, Francisco C.

AU - Stolyarova, Anna

AU - Barakat, David J.

AU - Bunge, Mary B

PY - 2004/12/1

Y1 - 2004/12/1

N2 - Microglia respond in a stereotypical pattern to a diverse array of pathological states. These changes are coupled to morphological and immunophenotypical alterations and the release of a variety of reactive species, trophic factors and cytokines that modify both microglia and their cellular environment. We examined whether a microglial-produced cytokine, tumour necrosis factor-α (TNF-α), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF-α antisense deoxyoligonucleotides (ASOs). Microglia and macrophages harvested from 3 d post-contused rat spinal cord were large and rounded (86.3 ± 9.6%). They were GSA-IB4-positive (GSA-IB4+) (Griffonia simplicifolia lectin, microglia specific; 94.8 ± 5.1%), strongly OX-42 positive (raised against a type 3 complement/integrin receptor, CD11b; 78.9 ± 9.1%), ED-1 positive (a lysosomal marker shown to correlate well with immune cell activation; 97.2 ± 2.6%) and IIA positive (antibody recognizes major histocompatibility complex II; 57.2 ± 5.6%), indicative of fully activated cells, for up to 48 h after plating. These cells also secreted significant amounts of TNF-α (up to 436 pg/μg total protein, 16 h). Fluoroscein isothiocyanate-labelled TNF-α ASOs (5, 50 and 200 nM) added to the culture medium were taken up very efficiently into the cells (> 90% cells) and significantly reduced TNF-α production by up to 92% (26.5 pg/μg total protein, 16 h, 200 nM TNF-α ASOs). Furthermore, few of the treated cells at this time were round (5.4 ± 2.7%), having become predominantly spindle shaped (74.9 ± 6.3%) or stellate (21.4 ± 2.7%); immunophenotypically, although all of them remained GSA-IB4 positive (91.6 ± 6.2%), many were weakly OX-42 positive and few expressed either ED-1 (12.9 ± 2.5%) or IIA (19.8 ± 7.4%). Thus, the secretion of TNF-α early in spinal cord injury may be involved in autoactivating microglia/macrophages. However, at the peak of microglial activation after injury, the activation state of microglia/macrophages is not stable and this process may still be reversible by blocking TNF-α.

AB - Microglia respond in a stereotypical pattern to a diverse array of pathological states. These changes are coupled to morphological and immunophenotypical alterations and the release of a variety of reactive species, trophic factors and cytokines that modify both microglia and their cellular environment. We examined whether a microglial-produced cytokine, tumour necrosis factor-α (TNF-α), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF-α antisense deoxyoligonucleotides (ASOs). Microglia and macrophages harvested from 3 d post-contused rat spinal cord were large and rounded (86.3 ± 9.6%). They were GSA-IB4-positive (GSA-IB4+) (Griffonia simplicifolia lectin, microglia specific; 94.8 ± 5.1%), strongly OX-42 positive (raised against a type 3 complement/integrin receptor, CD11b; 78.9 ± 9.1%), ED-1 positive (a lysosomal marker shown to correlate well with immune cell activation; 97.2 ± 2.6%) and IIA positive (antibody recognizes major histocompatibility complex II; 57.2 ± 5.6%), indicative of fully activated cells, for up to 48 h after plating. These cells also secreted significant amounts of TNF-α (up to 436 pg/μg total protein, 16 h). Fluoroscein isothiocyanate-labelled TNF-α ASOs (5, 50 and 200 nM) added to the culture medium were taken up very efficiently into the cells (> 90% cells) and significantly reduced TNF-α production by up to 92% (26.5 pg/μg total protein, 16 h, 200 nM TNF-α ASOs). Furthermore, few of the treated cells at this time were round (5.4 ± 2.7%), having become predominantly spindle shaped (74.9 ± 6.3%) or stellate (21.4 ± 2.7%); immunophenotypically, although all of them remained GSA-IB4 positive (91.6 ± 6.2%), many were weakly OX-42 positive and few expressed either ED-1 (12.9 ± 2.5%) or IIA (19.8 ± 7.4%). Thus, the secretion of TNF-α early in spinal cord injury may be involved in autoactivating microglia/macrophages. However, at the peak of microglial activation after injury, the activation state of microglia/macrophages is not stable and this process may still be reversible by blocking TNF-α.

KW - Cytokines

KW - Gene therapy

KW - Inflammation

KW - Macrophage

KW - Microglia

KW - Rodent

UR - http://www.scopus.com/inward/record.url?scp=10944236178&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=10944236178&partnerID=8YFLogxK

U2 - 10.1111/j.1460-9568.2004.03799.x

DO - 10.1111/j.1460-9568.2004.03799.x

M3 - Article

C2 - 15610171

AN - SCOPUS:10944236178

VL - 20

SP - 3387

EP - 3396

JO - European Journal of Neuroscience

JF - European Journal of Neuroscience

SN - 0953-816X

IS - 12

ER -