Microglial cells (MC) are the brain-resident immune cells. After acute activation, as a result of neuronal injury, MC proliferates, change their morphology (reactive microgliosis) and secrete neurotoxic factors. The activities of these cells are mostly beneficial, becoming destructive only when they escape from the strict control normally made by the Central Nervous System (CNS) envirnment. Downregulation of inflammatory mediators and removal of activated microglia may be key underlying mechanisms by which brain inflammation is controlled. Furthermore, anti-inflammatory cytokines, such as IL-4 and IL-13, may participate in these regulatory mechanisms present in the CNS. Therefore, we evaluated the effects of IL-4 on MC survival.
Here, we show that IL4 induced a significant increase in the apoptosis of N9 microglial cells after 48 h of culture (hypodiploid DNA content, p = 0.0001). IL4 also increased the percentage of annexin-V single positive and annexin-V/7AAD double positive cells, indicating induction of early and late apoptosis respectively. In addition, we found that IL4 significantly increased the cleavage of caspase 3 (p <0.01). Moreover, the IL-4-induced cell death was blocked by the presence of a clasical pan caspase inhibitor zVAD (p <0.05). We also determined that caspase 3 was not only cleaved but also activated, by detection of cleaved PARP in IL-4-stimulated cells. PARP is one of the specific substrates of caspase 3 and a critical factor involved in DNA repair to prevent apoptosis. In another set of experiments we observed that IL-4 was able to reduce the levels of Bcl-XL (western blot) and constitutive authophagy (microscopy, MDC staining), two factors related to cell survival.
Our preliminary results suggest that induction of MC death may be an additional mechanism induced by IL4 to control the extent and duration of neuroinflammation.
