We have previously shown that recombinant LIF protects neurons against glutamate-induced excitotoxicity . In this study, we investigated the mechanism by which astrocytes produce and release LIF. Here we show that glutamate-induced neuronal excitotoxicity leads to adenosine receptor-mediated increase in LIF mRNA expression in cultured cortical astrocytes. We demonstrate that the upregulation of LIF mRNA and protein is adenosine A2B receptor-dependent, and is mediated through Gq/11-PLC-PKC-MAPK-NF-κB signaling pathways. We furthermore show that LIF is transiting through the Golgi and is found in recycling endosomes rather than in LDCV. Finally, LIF produced by astrocytes can protect neurons against excitotoxicity.
It has been known for more than a decade that astrocytes are the major source for LIF in the CNS [18, 19, 59, 60]. However, the factors responsible for the regulation of LIF expression in these cells are still largely unknown. It is well known that stressed neurons release nucleotides such as ATP and adenosine [30, 61]. Recently, it was demonstrated that astrocytes increase LIF production and release in response to ATP receptor stimulation . In this study, the authors demonstrate that neurons during action potentials can secrete ATP, which triggers LIF production in astrocytes. This ATP-dependent upregulation of LIF by astrocytes is responsible for the promotion of oligodendrocyte-mediated myelination around neuronal axons. ATP is also known to be secreted by neurons during stressful conditions such as seizure, ischemia and hypoxia [26, 27]. However, when we blocked adenosine receptors with the non-selective antagonist caffeine, or with specific A2A/A2B receptor antagonists, the effect of glutamate-stressed neuronal supernatants on LIF expression in astrocytes was absent, suggesting that adenosine, but not ATP, is responsible for astrocytic LIF production during glutamate-induced neuronal stress. Thus, it might be hypothesized that depending on the CNS status, astrocytic LIF expression and secretion is differentially regulated; during normal neuronal activity and development ATP is involved whereas during neuronal insults, adenosine might enhance LIF secretion by astrocytes.
Several studies have demonstrated the involvement of adenosine A2B receptors in the regulation of IL-6 expression in various cell types in vitro[38, 47, 48, 62, 63] as well as in vivo, suggesting that A2B receptors might also be essential in the regulation of other IL-6-type cytokines. Our results show that adenosine-dependent LIF regulation is mediated through the A2B receptor, since no increase in LIF expression was found in cultured astrocytes from A2B receptor deficient mice. Instead NECA caused a down-regulation of LIF mRNA after 8 and 24 hours in these cells, indicating that knocking out A2B receptors may have unmasked an inhibitory effect on LIF mRNA expression of an unidentified adenosine receptor. Whether or not this might explain the very short-lived effect of NECA on LIF mRNA expression in wild-type astrocytes is at the moment unclear and a subject of future investigations. We furthermore demonstrated that A2B-mediated LIF expression is dependent on the PKC, but not the PKA pathway. These data are in line with the study of Aloisi and colleagues, which demonstrated that LIF modulation by pro-inflammatory cytokines in human astrocytes was mediated through PKC activation . Moreover, PKC has also been shown to be essential in IL-6 regulation [47, 48, 62, 65], revealing a prominent role for PKC in the signaling pathway controlling LIF gene expression.
MAPKs have been reported to be involved in adenosine A2B receptor-mediated regulation of IL-6 gene expression in astrocytoma cells . In our experiments, both basal as well as NECA-induced LIF gene expression and release in cultured astrocytes were inhibited by specific inhibitors of p38 and ERK1/2, but not JNK-MAPKs. In line with our findings, it has been shown that LIF expression in Schwann cells is mediated through PKC pathway-induced ERK1/2 activation . Furthermore, we show here that adenosine-dependent LIF expression in astrocytes is regulated through the NF-κB transcription factor. This observation is in line with several studies showing an NF-κB-dependent regulation of IL-6 gene by this transcription factor in several cell types [38, 50, 51, 65, 66]. It has been shown that NECA-induced NF-κB activation and the resultant IL-6 gene expression was abolished by inhibitors of MAPK pathways . In our study, preliminary observations indicate that NECA-induced activation of the NF-κB pathway is reduced by selective inhibitors of p38 and ERK1/2 pathways (data not shown), suggesting that these pathways might play as upstream mediators in NF-κB-dependent LIF expression in astrocytes.
Recent evidence indicates that, depending on the cell type, different secretory pathways are employed for cytokine release . For example, T cells use two different release mechanisms: IL-2 and IFN-γ are secreted at the immunological synapse whereas CCL3 and TNF-α are secreted multidirectionally, suggesting different secretory pathways . In neurons or neuron-like cells, secretory granules called LDCVs are the organelles used for the selective secretion of IL-6, TGF-β2 and CCL21 [53, 54, 69]. The same organelles are also used in immune cells such as mast cells and neutrophils . Here we show that LIF protein is transported through Golgi but its secretion by astrocytes is not mediated by secretory granules. Instead, LIF co-localizes with Rab11, a known marker of recycling endosomes [57, 58]. Moreover, we observed a partial co-localization of LIF with clathrin, which also associates with recycling endosomes where it is implicated in protein sorting . Recycling endosomes have now been shown to be responsible for cytokine secretion in several cell types. For example, IFN-γ and TNF-α secretion from natural killer cells require Rab11 . Recycling endosomes are also responsible for the constitutive secretion of IL-6 and TNF-α in macrophages . Further studies will be needed to better understand LIF sorting, trafficking and release by these vesicles.
Interestingly, our data indicate that LIF is constitutively released from astrocytes. Indeed constant levels of LIF were present in the supernatants of untreated astrocytes when measured by ELISA. Similar data were observed in human astrocyte cultures . Whether this observation is representative of the physiological behavior of astrocytes in vivo or is due to the culture conditions remains to be determined. We further show that by blocking the early secretory pathway with BFA, the LIF concentration in the culture supernatant was not increased upon NECA stimulation. The inhibitory effect of BFA indicates that LIF passes through the Golgi prior to its secretion, and thus does not follow non-conventional secretory pathways that by-pass the Golgi and is typically insensitive to BFA, which has recently been reported to be used by other cytokines . Importantly, the inhibitory effect of BFA suggests that NECA-stimulated release of LIF by astrocytes requires de novo LIF synthesis, and does not involve a ready-releasable post-Golgi pool of LIF.
It is now clear that one of the major roles of LIF is directed toward cell protection. Indeed, it has been shown that LIF is up regulated in astrocytes and neurons after cerebral ischemia  as well as in astrocytes after cortical brain injury , suggesting a role of LIF in neuronal repair or protection. In line with these data, treatment of rat with LIF prevented loss of motoneurons after peripheral nerve injury [73, 74] and protection of retinal ganglia cells was compromised in LIF knock-out mice after lens injury . Finally, LIF was shown to limit demyelination in an experimental autoimmune encephalomyelitis mouse model  and has become a prominent therapeutic candidate for multiple sclerosis . We have previously shown that LIF can protect cortical as well as hippocampal neurons against glutamate-induced excitotoxicity . Here we show that LIF coming from the supernatant of NECA-treated astrocytes has the same protective effect. Indeed, astrocytes produce several other cytokines and neurotrophic factors including IL-6, NGF, brain-derived neurotrophic factor, neurotrophin-3, S-100β protein and TGFβ , that might help neurons to cope with excitotoxic stress. Accordingly, conditioned media from astrocyte cultures protected cortical neurons against glutamate (data not shown). In order to confine the neuroprotective effect of astrocytic factors that are released in response to NECA treatment, we had to refresh astrocyte culture medium prior to NECA treatment and testing supernatant on glutamate-stressed neurons. This, together with LIF neutralization, indicates that LIF produced by astrocytes after adenosine receptor stimulation is necessary to witness neuronal protection. Our results provide further evidence for a role of adenosine in neuronal protection. Indeed, it has been shown that adenosine can protect neurons during hypoxia [76, 77], ischemia [78, 79] and excessive neuronal activity [80, 81]. This adenosine protection is often mediated through the A1 receptor subtype [82–84], but here we show that an indirect protection of adenosine through the stimulation of A2B receptor on astrocytes leading to LIF upregulation exists. This A2B receptor activation might be related to an anti-inflammatory process as observed previously by others [85–87].