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

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-alpha (TNF-alpha), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF-alpha 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-alpha (up to 436 pg/microg total protein, 16 h). Fluoroscein isothiocyanate-labelled TNF-alpha 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-alpha production by up to 92% (26.5 pg/microg total protein, 16 h, 200 nm TNF-alpha 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-alpha 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-alpha