Abstract
Purpose: :
Human optic nerve astrocytes (HONA) are the major glial cell type in the optic nerve head region. HONA provide metabolic and homeostatic support to the ganglion cell axons. Under pathological conditions such as glaucomatous neuropathy astrocytes become reactive and release inflammatory mediators including tumor necrosis factor-alpha (TNF-α), express oxidative markers such as nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and behave as antigen-presenting cells. This study describes the effect of pro-inflammatory mediators on cytokine expression by HONA in an in vitro model of glaucomatous optic neuropathy.
Methods: :
Cultured HONA (from two male donors, 19 and 55 years old with no history of ocular disease), characterized as 99% glial fibrillary acidic protein (GFAP)-positive cells, were used in this study. We exposed confluent cultures of HONA for 24 hours to bacterial endotoxin lipopolysaccharide (LPS; 10 µg/mL), interleukin 1-beta (IL-1ß; 10 ng/mL), and TNF-α (10 ng/mL), with or without the corticosteroid triamcinolone acetonide (TA; 10 ng/mL) or the specific inhibitor of iNOS aminoguanidine (AG, 10 µM). Inflammatory cytokines (Interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), and Regulated upon Activation, Normal T-cell Expressed and Secreted (RANTES)) released into the conditioned media (CM) were assayed using ELISA and a multiplex platform (Luminex). NO was also assessed in the CM by fluorometric measurement of total nitrate/nitrite levels. iNOS was detected by PAGE-Western blot.
Results: :
Increases in chemokines IL-6, IL-8, MCP-1 and RANTES were observed in CM of HONA treated with LPS, IL-1ß, and TNF-α, compared to control cultures. The concurrent addition of TA but not GA significantly reduced the expression of cytokines induced by LPS, IL-1ß, and TNF-α treatment. No differences in cytokine expression were observed between the two HONA donors. There was good correlation between ELISA and Luminex results for cytokine expression. Treatments with pro-inflammatory mediators for 24 hours did not induce an increase in released NO.
Conclusions: :
HONA respond to pro-inflammatory stimuli by expressing a range of immune mediators, such as acute phase cytokines IL-6, IL-8, MCP-1 and RANTES. Drugs that modulate theses molecules as well as NO and iNOS in HONA will have therapeutic value as neuroprotective agents for glaucomatous and inflammatory ocular diseases.
Keywords: optic nerve • cytokines/chemokines • inflammation