- Top of page
- Materials and methods
Microglia-mediated cytotoxicity has been implicated in models of neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease, but few studies have documented how neuroprotective signals might mitigate such cytotoxicity. To explore the neuroprotective mechanism of anti-inflammatory cytokines, we applied interleukin-4 (IL-4) to primary microglial cultures activated by lipopolysaccharide as well as to activated microglia cocultured with primary motoneurons. lipopolysaccharide increased nitric oxide and superoxide (O2·−) and decreased insulin-like growth factor-1 (IGF-1) release from microglial cultures, and induced motoneuron injury in microglia-motoneuron cocultures. However, lipopolysaccharide had minimal effects on isolated motoneuron cultures. IL−4 interaction with microglial IL-4 receptors suppressed and nitric oxide release, and lessened lipopolysaccharide-induced microglia-mediated motoneuron injury. The extent of nitric oxide suppression correlated directly with the extent of motoneuron survival. Although IL-4 enhanced release of free IGF-1 from microglia in the absence of lipopolysaccharide, it did not enhance free IGF-1 release in the presence of lipopolysaccharide. These data suggest that IL-4 may provide a significant immunomodulatory signal which can protect against microglia-mediated neurotoxicity by suppressing the production and release of free radicals.
An increasing number of studies suggest that neuronal death in neurodegenerative diseases is non-cell autonomous, and may involve non-neuronal cells. The potential contribution of non-neuronal cells to motoneuron injury and death is supported by the fact that selective overexpression of the mSOD1 transgene in neurons is not sufficient to cause motoneuron disease in mice (Pramatarova et al. 2001; Lino et al. 2002). A definitive study with chimeric mice demonstrated that normal motoneurons surrounded by mSOD1-expressing glia showed signs of injury, whereas mSOD1-expressing neurons surrounded by normal glia remained intact (Clement et al. 2003). Microglia, as resident immunocompetent cells within the CNS, play an important role in the progression of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease (for review, see Block and Hong 2005; Sargsyan et al. 2005). Microglial activation has been shown to parallel neuronal degeneration (Alexianu et al. 2001; Henkel et al. 2006). The question to be raised was whether microglial-mediated cytotoxicity can be abrogated, and by what signals.
T cells have been demonstrated to reduce neuronal injury in disease models of Parkinson's disease, ALS and Alzheimer's disease (Town et al. 2002; Angelov et al. 2003; Schwartz et al. 2003; Benner et al. 2004). However, fewer studies have clearly documented what specific signals from T cells convey neuroprotection in motoneuron injury paradigms. We hypothesized that the Th2 lymphocyte-derived anti-inflammatory cytokine interleukin-4 (IL-4) may be one of the potential neuroprotective signals. IL-4 is an important immunosuppressive mediator in the CNS. It modulates major histocompatibility complex class II expression and suppresses nitric oxide by microglia (Butovsky et al. 2005; Suzumura et al. 1994; Ledeboer et al. 2000). IL-4 has also been shown to reduce the production of pro-inflammatory cytokines such as IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), MIP-1α and RANTES in glial cultures treated with lipopolysaccharide or IL-1 (Ledeboer et al. 2000; Ehrlich et al. 1998). Recently, it has been documented that protection of IL-4 was attributed to down-regulation of TNF-α and up-regulation of insulin-like growth factor 1 (IGF-1) from microglia (Butovsky et al. 2005, 2006).
Our previous studies have documented the neurocytotoxic potential of activated microglia mediated by the release of nitric oxide (nitric oxide), superoxide (), and hydrogen peroxide (H2O2) (Le et al. 2001; Zhao et al. 2004). In the present study, we examined the effects of IL-4 in primary microglia cultures and in microglia cocultured with primary motoneurons. To mimic microglial activation in neurodegenerative diseases, lipopolysaccharide, a widely accepted triggering agent to microglia, was used. Following activation with lipopolysaccharide, microglia were noted to release more nitric oxide and , and lower levels of free IGF-1, an active form of the neurotrophic factor. The addition of IL-4 protected against the activated microglia-mediated motoneuron injury by reducing nitric oxide production and suppressing microglial production. This anti-inflammatory cytokine also enhanced free IGF-1 without lipopolysaccharide stimulation. However, with lipopolysaccharide, there was a minimal increase in free IGF-1, yet the cytokine was still neuroprotective.
- Top of page
- Materials and methods
There is growing evidence that activation of microglia contributes to pathophysiologic changes seen in various neurological disorders due to the generation of several substances implicated in neurotoxicity, including reactive oxygen species (ROS), such as nitric oxide and superoxide (); pro-inflammatory, neurotoxic cytokines, such as TNF-α; and glutamate (Gonzalez-Scarano and Baltuch 1999; Bal-Price and Brown 2001; Barger and Basile 2001; Le et al. 2001; Pocock and Liddle 2001; Zhao et al. 2004; Floden et al. 2005). The present study demonstrates that the anti-inflammatory cytokine IL-4 can protect motoneurons from injury induced by lipopolysaccharide-activated microglia. The neuroprotective effect is primarily mediated by reducing nitric oxide production and inhibiting release from activated microglia. To our knowledge, this is the first report that IL-4 has beneficial effects on motoneuron injury mediated by microglia.
In our previous study, we demonstrated that special iNOS inhibitor, L-NIL, significantly reduced nitric oxide production and reversed motoneuron death mediated by activated microglia, suggesting nitric oxide is an important contributor to neurotoxicity in our in vitro system (Zhao et al. 2004). Our present results reveal a strong negative correlation between nitric oxide levels (measured by nitrite + nitrate levels) and motoneuron survival in cocultures treated with IL-4 and exogenously added nitric oxide reversed neuroprotection of IL-4. These suggest that nitric oxide is a key factor modulated by IL-4 in our cell culture system. Nitric oxide is synthesized by iNOS in microglia. iNOS protein expression was down-regulated by IL-4 in activated microglia when IL-4 was given either before or after the triggering signal, lipopolysaccharide. More strikingly, we found that IL-4 increased motoneuron survival to a greater extent when added to MN + Mc cocultures after lipopolysaccharide than when added prior to lipopolysaccharide. In an effort to investigate the cause of this increase, we examined the levels of IL-4 mRNA. We detected increased IL-4R mRNA levels after microglia were activated with lipopolysaccharide. Thus, the greater expression of IL-4R with the subsequent greater efficacy of the available IL-4 could provide one potential explanation of the increased suppressive effects of IL-4 on iNOS expression in activated microglia. Two studies have reported the inhibitory effects of IL-4 on iNOS in lipopolysaccharide-stimulated astrocytes and mixed glial cultures (Brodie et al. 1998; Kitamura et al. 2000), although in their studies the cells were all treated with IL-4 first and then stimulated cell with other triggering signals.
Floden et al. (2005) demonstrated that Aβ-stimulated microglia released TNF-α and glutamate, which synergistically increased NOS expression and activity in neurons by western blot and immunocytochemistry. In our previous paper (Zhao et al. 2004) we also showed that lipopolysaccharide induced microglia to release TNF-α and higher levels of glutamate (30–40 μm) present in the MN and Mc cultures. Therefore, in addition to microglia iNOS expression, neuronal NOS may be another source of nitric oxide contributing to motoneuron death in our in vitro system. It has also been shown that TNF-α had neurotoxic effects through up-regulating iNOS, nitric oxide and production from microglia (Kuno et al. 2005). It has also been documented that neuroprotection of IL-4 was attributed to down-regulation of TNF-α (Butovsky et al. 2005). In accordance with this, we did find lipopolysaccharide dramatically enhanced TNF-α produced by microglia (Zhao et al. 2004) and IL-4 significantly decreased TNF-α levels in MN + Mc + lipopolysaccharide cocultures (data not shown). Therefore, down-regulation of TNF-α is one of mechanisms by which IL-4 inhibited nitric oxide and production and protected motoneurons from microglia-mediated toxicity.
Another finding in the current study is that IL-4 increased free IGF-1, the active form of this neuroprotective factor, in untreated microglia cultures and that lipopolysaccharide decreased free IGF-1. At first, we hypothesized that the induction of free IGF-1 in microglia might be another aspect of IL-4 neuroprotection; however, our subsequent results suggested it might not be. IL-4 increased motoneuron survival in both lipopolysaccharide-treated MN + Mc cocultures; however, IL-4 did not increase free IGF-1 levels in the same cocultures. In addition, we saw no increase in IGF-1 mRNA in these lipopolysaccharide-activated cultures, although it has been reported that IL-4 up-regulated IGF-1 mRNA in both untreated and lipopolysaccharide-activated microglia (Butovsky et al. 2005). Additionally, even though free IGF-1 was up-regulated by IL-4 in untreated microglia cocultured with motoneurons, IL-4 did not increase motoneuron survival under the same conditions. Finally, we also found a poor correlation between free IGF-1 levels and motoneuron survival in MN + Mc cocultures. Therefore, these results suggest that IGF-1 may not be the primary mechanism of IL-4 neuroprotection. However, we should clarify three points. Firstly, IGF-1 is clearly a generally neuroprotective trophic factor that has been shown to delay disease progression in several neurodegenerative models (Dore et al. 1999; Kaspar et al. 2003). Although we cannot discount the possibility that there may have been an immediate local increase in IGF-1 that was quickly sequestered by binding proteins, it is likely that the free IGF-1 levels induced by IL-4 either before or after lipopolysaccharide in our system were not sufficient to elicit the neuroprotective effects. Secondly, because free IGF-1 levels are determined by total IGF-1 and the levels of the five known binding proteins (Denley et al. 2005), there may have been increases in both IGF-1 and one or more of the binding proteins that resulted in no net change in free IGF-1. Finally, it is possible that neurotrophic factors other than IGF-1 may enhance the neuroprotective actions of this anti-inflammatory cytokine.
Several studies have demonstrated the beneficial effects of IL-4 in the CNS. Chao et al. (1993) reported that IL-4 blocked microglia-mediated cerebral neuron injury. It has also been reported that IL-4 increased neuronal survival in hippocampal mixed cultures (Araujo and Cotman 1993). IL-4 delivered by transfected cells inhibited the progression and the severity of experimental autoimmune encephalomyelitis (Shaw et al. 1997; Furlan et al. 2001). Furthermore, gene transfer of IL-4 to the retina enhanced the survival of axotomized retinal ganglion cells (Koeberle et al. 2004). While these studies demonstrate that IL-4 can be neuroprotective, few studies have examined the mechanisms of neuroprotection. Our present study provides evidence that IL-4 protects motoneurons from injury partly by decreasing free radicals released from microglia and may not involve free IGF-1. Whereas one paper reported that IL-4 did not significantly suppress nitric oxide production from microglia when given 24 h after the triggering signals IFN-γ and lipopolysaccharide (Chao et al. 1993), we found that IL-4 was more neuroprotective when added 2 h after lipopolysaccharide than before lipopolysaccharide. These data suggest that anti-inflammatory intervention at the earliest possible time would be essential to achieve meaningful neuroprotection in vivo.
Besides IL-4, IL-10 and transforming growth factor-beta (TGF-β) are two other major anti-inflammatory cytokines. We had detected the effects of IL-10 and TGF-β in our MN + Mc coculture system as well as IL-4. We did not observe a neuroprotective effect of TGF-β in the cultures (data not shown). We found that IL-4 had stronger protective effects on motoneurons than IL-10. Like IL-4, IL-10 inhibited iNOS expression and nitric oxide production from microglia (data not shown). However, IL-10 increased microglial release, and also decreased rather than increased IGF-1 release from microglia (data not shown). Further studies are clearly necessary to define the mechanism of IL-10 effects.
One potential source of the neuroprotective signal, IL-4, is Th2 lymphocytes. Recent in vivo studies have shown that T-cell immunomodulation may be neuroprotective in models of neurodegenerative diseases. Angelov et al. (2003) reported that glatiramer acetate, a polypeptide which can activate a wide range of self-reactive T cells, was neuroprotective in a transgenic model of ALS. In a Parkinson's disease model, adoptive transfer of splenocytes from mice immunized with glatiramer acetate significantly protected nigrostriatal neurons against MPTP-induced neurodegeneration (Benner et al. 2004). Furthermore, CD4+ T cells have been documented to enhance facial motoneuron survival after facial nerve injury (Byram et al. 2004; Jones et al. 2005). However, these studies did not examine which protective signals are involved in the beneficial mechanisms in the neurodegenerative models. Our present data suggest that IL-4 may be one of the significant signals in T-cell-mediated neuroprotection. Accordingly, immunological strategies to increase the presence of Th2 lymphocytes and the release of IL-4 would be a promising direction for treatment of neurodegenerative disorders. In the present study, the results are related to the effects of lipopolysaccharide, but most stress conditions in neurodegenerative diseases are not due to the presence of lipopolysaccharide. We could not exclude that different subsets of microglia cells activated by other stimuli might be involved in neurodegenerative diseases. In vivo studies will clearly be required to determine whether IL-4 achieves neuroprotection by mitigating the neurotoxic effects of microglia.