A water-soluble carbon monoxide-releasing molecules (CORM-3) reduces neuro-inflammation in BV-2 microglia

Mohamed Bani-Hani1,2, David Greenstein2, Brian E. Mann3, Colin J. Green1, Roberto Motterlini1. 1Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow; 2North west London Hospitals NHS Trust, Northwick Park Hospital, Harrow; 3Department of Chemistry, University of Sheffield, Sheffield, United Kingdom.

Microglial cells play a major role in regulating the neuronal inflammatory response, but their excessive stimulation is blamed for neuronal injury in various neurodegenerative diseases. Interferon-γ (INFγ) is one of the most potent microglial stimuli (Popko B et al., 1997) that act synergistically with other pro-inflammatory cytokines in driving multiple destructive cascades. Carbon monoxide-releasing molecules (CO-RMs) are emerging as a new class of pharmacological agents that modulate important cellular function by liberating CO in biological system. Our group has developed a water-soluble CO releaser (CORM-3) which showed remarkable cardioprotective and anti-inflammatory actions (Clark JE 2003 et al.; Sawle P et al., 2005). In the present study we examined the effect of CORM-3 on INFγ-induced inflammation in BV-2 microglial cells under normoxic and hypoxic conditions. BV-2 microglial cells were incubated with INFγ (15 ng/ml) for 24 h in normoxic conditions or subjected to 12 h hypoxia and reoxygenation (24 h) in the presence of INFγ. In normoxia, INFγ caused an increase in NO release (nitrite levels) from 0.09±0.059 to 36.6±4.5μM (p<0.001) (n=12) and an increase in TNF-α production from 162±5.7 to 405±44 pg/ml (p<0.001) (n=10). Similarly, after hypoxia-reoxygenation (H/R) INFγ increased nitrite levels from 0.8±0.18 to74.5±1.3 μM and increased TNF-α production from 101.8±4 to 293±6 pg/ml (p<0.01) the apparent reduction in TNF-α production is due to the high level of cellular injury (90% of cells) resulting from the combination of H/R and INFγ . CORM-3 (75 μM) significantly attenuated INFγ-mediated increase in nitrite levels in normoxia (from 36.6±4.5 to 5.5±0.9 μM) (P<0.001) and in (H/R) from 74.5±1.3 to 59.7± 1μM(p<0.001). In addition, CORM-3 reduced TNF-α production in normoxia from 405±44 to 206±14 pg/ml (p<0.001), and in H/R from 293±6 to198±21 (p<0.001). An inactive compound, which does not liberate CO (iCORM-3), failed to prevent the increase in inflammatory mediators mediated by INFγ suggesting that CO is responsible for the observed effects. Furthermore, blockade of endogenous heme oxygenase-derived CO by tin protoporphyrin-IX (10 μM) did not affect CORM-3 induced anti-inflammatory activity. CORM-3 did not show any cytotoxicity and appears to exert its effect at multiple levels through interaction with heme-dependent pro-inflammatory enzymes (NAD(P)H-oxidase and iNOS) and various signal transduction pathways (MAPK, P38, JNK, PI3K). These results suggest that CORM-3 could be used to modulate microglial activity in neuro-inflammatory diseases.

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Sawle P. et al.Br. J. Pharmacol. 2005; 145:800-10.