The findings of the present study indicate that IGF-I increases the expression of IL6, IL-1β and TNF-α in cultured astrocytes under basal conditions. In contrast, IGF-I decreases expression of IL6 and IL-1β in cultured astrocytes submitted to a proinflammatory challenge by treatment with LPS. Under basal conditions, the effects of IGF-I on expression of IL6, IL-1β and TNF-α were dependent on IGF-I dose, promoting these cytokine expressions at the lower dose. When challenged with LPS, however, IGF-I at both dosages exerted similar inhibitory effects on the expression of IL6 and IL-1β.
The increases in IL6, IL-1β and TNF-α mRNA levels detected in astrocytes after treatment with IGF-I under basal conditions does not necessarily imply a proinflammatory action of IGF-I. Cytokines such as IL6, IL-1β and TNF-α have different physiological functions, including regulation of neuronal development, ionic homeostasis, neuropeptide release and synaptic plasticity [31–34]. Therefore, the observed increases in IL6, IL-1β and TNF-α levels under basal conditions may represent a physiological action of IGF-I. In addition, IGF-I reduced expression of TLR4 in both the presence and absence of LPS. TLR4 is a member of the IL-1receptor/TLR superfamily that is expressed by astrocytes and that is required for an LPS-induced inflammatory response [35–38]. Therefore the observed decrease in TLR4 expression by IGF-I under basal conditions and after LPS proinflammatory challenge may contribute to a reduced capacity of astrocytes to be activated by proinflammatory molecules such as LPS. Indeed, IGF-I reduced the effects of LPS on mRNA levels of IL6, and IL-1β in astrocytes, in agreement with previous findings showing that IGF-I down-regulates cytokine expression induced by LPS in adult mouse brain .
Having established that exogenous IGF-I regulates the expressions of IL6, IL-1β, TNF-α and TLR4 in astrocytes, we then assessed whether an increase in endogenous production of IGF-I by astrocytes might also affect expression of these molecules. The findings of the present study indicate that a recombinant adenoviral vector harboring the gene for rat IGF-I (RAd-IGF-I) is able to increase the production of IGF-I in astrocytes, without affecting IGFBP2 or IGF-I receptor levels. Under basal conditions, RAd-IGF-I resulted in an effect similar to that of IGF-I, increasing the expression of IL6 and IL-1β and decreasing the expression of TLR4. Adenoviral vector infection per se also had an effect, since mRNA levels of IL6 and IL-1β were increased in astrocytes 72 h after incubation with control vector, RAd-TK/GFP. In addition, TNF-α mRNA levels were increased in astrocytes incubated for 72 h with RAd-IGF-I. However, no increase for IL6 or IL-1β was detected at 72 h in astrocytes infected with RAd-IGF-I, suggesting that IGF-I is compensating for some of the proinflammatory actions of the adenoviral vector. Indeed, RAd-IGF-I exerted a clear anti-inflammatory action on LPS-treated astrocytes, decreasing the mRNA levels of IL6, IL-1β, TNF-α and TLR4. Therefore, RAd-IGF-I imitated the effects of IGF-I on LPS-stimulated astrocytes. These findings demonstrate efficacy of IGF-I gene therapy in reducing the response of astrocytes to an inflammatory challenge. However, as was observed for exogenous IGF-I, the anti-inflammatory action of IGF-I gene therapy was transient, since RAd-IGF-I did not significantly affect levels of IL6, IL-1β, or TNF-α in cultures incubated with the viral vector for 72 h.
Our data suggest that RAd-IGF-I, like IGF-I, reduces the effects of LPS on astrocytes by decreasing the expression of TLR4. Activation of TLR4 results in translocation of the NFκB p65 subunit to the cell nucleus and consequent activation of NFκB-mediated transcription of proinflammatory cytokines and chemokines [40–44]. In agreement with the effect of RAd-IGF-I on TLR4 expression, and with the results of a previous study on the effect of IGF-I on astrocytes , IGF-I gene therapy prevented LPS-induced translocation of the NFκB p65 subunit to the cell nucleus. This suggests that RAd-IGF-I exerts, at least in part, its anti-inflammatory action by downregulation of TLR4 and subsequent inhibition of NFκB activity.
The anti-inflammatory action of RAd-IGF-I in LPS-stimulated astrocytes may be mediated by IGF-I receptor activation, since it was prevented by incubation of astrocytes with cyclolignan picropodophyllin (PPP), an inhibitor of the IGF-I receptor tyrosine phosphorylation [45–47]. However, PPP treatment per se increased IL6 mRNA levels in cells infected with Rad-IGF-I construct or control construct under basal conditions, and increased mRNA levels of IL6 and IL-1β in LPS-treated astrocytes incubated with RAd-TK/GFP or RAd-IGF-I. These actions of PPP cannot be explained simply by IGF-I receptor inhibition, suggesting that PPP may have other effects under the experimental conditions used.