The principle findings of this study are that signaling via the IL-1R1 is not critical for the death of dentate granule neurons or the activation of microglia following chemical-induced injury. The data support previous observations of an attenuated astrocytic response to insult in mice deficient for IL-1R1 (Lin et al., 2006
). In addition, the study offers new data regarding the induction of components of the Fas/FasL (CD95/CD95L) signaling pathway upon injury in IL-1R1−/− mice.
A role of IL-1 in mediating brain injury has been previously suggested. For example, diminishing actions of IL-1 by either IL-1ra or deletion of IL-1 confers a level of protection in distinct models of brain injury (Garcia et al., 1995
; Loddick et al., 1997
; Boutin et al., 2001
). The recent work of Pinteaux et al., (2006)
suggests that the neuroprotection offered by IL-1ra is due to its production and release by microglial cells (Eriksson et al., 2000
). In the current study, the elevation in TNFα, TNFR1, and IL-1ra at 24 hrs suggests the induction of a neuroinflammatory response with this chemical induced hippocampal injury. While not elevated at 24 hrs, both IL-1α and IL-1β mRNA and protein levels are elevated later in the injury response corresponding to the prominent microglia phagocytosis (Bruccoleri et al., 1998
; Fiedorowicz et al., 2001
). An elevation of IL-1ra in the absence of an increase in IL-1 is consistent with the findings of Gabellec et al. (1999)
, which demonstrated a transient down regulation of receptor density and an elevation in IL-1ra in the early stages of traumatic injury. The limited induction of IL-1α and IL-1β mRNA, coupled with a lack of increase in IL-1R1 or MyD88 mRNA, at 24 hrs, along with the absence of any attenuation of the damage in IL-1R1−/− mice at 24 or 72 hrs, supports the minimal involvement of the receptor in regulating damage in the dentate granule neurons as induced by TMT.
While IL-1 shows neurotoxic actions, studies suggest that the protein does not directly initiate brain damage but rather exacerbates induced damage (Davies et al., 1999
). Consistent with the neuronal data from the current study, Touzani et al. (2002)
showed that, while the lack of both IL-1α and IL-1β provided neuroprotection (Boutin et al., 2001
), the absence of IL-1R1 was not sufficient to significantly inhibit the extent of injury from middle cerebral artery occlusion in mice. However, in both wildtype and IL-1R1−/− mice, the addition of IL-1 exacerbated the response. This exacerbation could be inhibited with the addition of IL-1ra in the wildtype mouse with no attenuation of the injury in the knockout mice. This data suggests that IL-1 could be acting through alternate receptor(s) in the absence of the type I receptor (Touzani, 2002
). A second receptor for IL-1 does exist; however, IL-1RII is considered a decoy receptor that binds IL-1 but fails to initiate signal transduction. In serving as a decoy, it can limit the biological activity of IL-1 (Colotta et al., 1994
). The previously reported localized up-regulation of IL-1R1 restricted to hippocampal regions of originating seizures suggests that a strong focal neuronal activation is required to upregulate this receptor in neurons (Ravizza and Vezzani, 2006
). However, this study also reported that TUNEL+ neurons were not the population of neurons expressing IL-1R1 following seizure. This would suggest a lack of association between neuron specific receptor activation and neuronal death under these conditions.
A decrease in neocortical microglia activation has been reported in IL-1R1−/− mice after hypoxia/ischemia (Basu et al., 2005
); however, whether this is due to a direct effect on the microglia or secondary to the diminished neuronal death is unknown. Given that the response of microglia cells is often linked to the level of cell damage within any specific brain region, evaluation and conclusions drawn regarding microglia actions can be confounded if neuronal/tissue damage is not equivalent for comparison. In the current study, a focal injury to dentate granule neurons was not altered with the absence of IL-1R1 and the response of microglia was equivalent to that seen in the wildtype mouse. In addition, the temporal and spatial progression of the microglia response followed a similar pattern for both groups. Within 24 hrs, pyknotic dentate granule cell neurons were present and this was accompanied by a response of microglia with a reactive phenotype. This response progressed to an activated phenotype and the phagocytosis of neuronal debris. Within each section and across groups, the relationship between the severity of neuronal death and microglia response was maintained, suggesting that the timing of reaction and functional role of the microglia is not altered in the absence of IL-1R1. This data suggests that IL-1R1 signaling is either not critical for this specific model of damage or more general, for dentate granule neurons. It also demonstrates that the microglia response is not regulated by IL-1R1 signaling given equivalent levels of neuronal damage.
While both the neuronal and microglia response were similar, the astrocyte response was very distinct between the two groups. Parker et al., (2002)
suggested that astrocytes are the primary target for IL-1 actions with IL-1R1-dependent activity demonstrated in reactive astrocytes. The expression of IL-1R1 has been linked to reactive astrocytes in a transient manner over 18–48 hrs post-seizure (Ravizza and Vezzanini, 2006
) and at 24 hrs following a puncture wound (Friedman, 2001
). Mice deficient for IL-1R1 displayed limited astrocytic processes within the dentate blades and a diminished GFAP induction as compared to wildtype mice in response to the injury process. Corresponding with the time at which IL-1 appears to be most influential in the TMT injury (Bruccoleri et al., 1998
; Fiedorowicz et al., 2001
), the most distinct differences in GFAP immunoreactivity between the groups occurred at 72 hrs. The diminished structural and GFAP response of astrocytes seen in the current study is similar to observations previously reported using a penetrating brain wound (Lin et al., 2006
). It is also consistent with observations that hippocampal astrocytes respond to IL-1 (Friedman et al., 2001
) and that astrocyte over-expression of IL-1R1 contributes to reactive astrogliosis (Giulian et al., 1988
). The specific elevation of TGFβ1 seen in IL-1R1−/− mice following TMT may contribute to the attenuated astrocytes response given its actions on astrocytes and role in scar formation (Lindholm et al., 1992
; Laping et al., 1994
). These immunosuppressive and wound healing actions of TGFβ1 are thought to control inflammation and to limit the extent of neuronal injury (Sei et al., 1995
While IL-1R1 mediates IL-1β inhibition of astrocytic glutamate re-uptake (Ye and Sontheimer, 1996
; Hu et al., 2000
), which could lead to a higher glutamate environment and subsequent neuronal death, the structural differences in astrocytes seen in the IL-1R1−/− mice has not necessarily been correlated with functional changes in glutamate uptake (Lin et al., 2006
). Stimulation of IL-1R1 can also induce the production of soluble factors such as, cytokines and growth factors (Croll et al., 2004
) and the activation of NFκB in astrocytes (Krohn et al., 1999
). These effects can be either beneficial or detrimental to neurons. A recent study by Thornton et al. (2006)
used primary cortical cell cultures to examine the influence of astrocytes in the IL-1β toxicity to neurons. The authors concluded that the activation of IL-1R1 in astrocytes and the release of free radicals induce a caspase-dependent death of neurons. This was supported by the in vivo
study of mild hypoxia/ischemia showing an attenuation of iNOS-mediated free radical damage in IL-1R1−/− mice (Lazovic et al., 2005
). In an acute brain injury, where the intervention of astrocytes to either provide growth factor support or to provide a physical glia barrier, a diminished astrocyte response may indeed be a critical factor in determining the severity of neuronal damage. However, in brain injury that targets a neuronal population directly, such as occurs in the TMT model and in targeted neuronal degenerative diseases, the involvement of astrocytes in the acute phase of neuronal death may not be as critical. Thus, the diminished response of astrocytes in the IL-1R1−/− mice would have little direct consequences on the severity of dentate granule cell death, yet would have a significant impact on a severe neuronal injury involving glia scarring.
The neurotoxicity of IL-1β has been linked to the upregulation of FasL in astrocytes (Ghorpade et al., 2003
) and the induction of neuronal apoptosis (Allan and Rothwell, 2001
; Deshpande et al., 2005
). While we found no protection from the hippocampal injury, the distinct changes seen in the IL-1R1−/− mice suggests an active role for the intracellular Fas signaling pathway that could contribute to the neuroprotection often seen in other models of brain injury. The lack of detectable FasL transcript levels would be consistent with the lack of a prominent astrocyte response at 24 hrs. Both FADD and FAP-1 are Fas receptor associated proteins and, as such, changes in gene expression may reflect the levels and activity of Fas. In the IL-1R1−/− mice, this pattern seems to hold with the lower basal transcript levels and the increase following TMT for all three transcripts. FADD is an adaptor protein for the extrinsic apoptotic receptor Fas, and is essential for multiple signaling events downstream of Fas (Juo et al., 1999
). FAP, however, is an anti-apoptotic tyrosine phosphatase that is located in the cytosol and serves as an intracellular regulator of Fas translocation to block the transduction of the death signal (Sato et al., 1995
). There is little data regarding the apoptotic signaling pathways recruited via IL-1 or in the absence of IL-1R1 activation; however, it is interesting to note that the changes in Fas-associated genes following TMT occurred exclusively in IL-1R1−/− animals. Although there are numerous mechanisms of signal transduction that are not reflected in transcript levels, such as protein phosphorylation, the fact that message levels for none of the components are modulated in the wildtype animals may suggest that this is not a primary mechanism of cell death in the TMT model. In addition, any activation of the Fas signaling pathway may not automatically be associated with the neuronal damage observed in this model. Up-regulation of FasL/CD95L has been attributed to astrocytic-induced elimination of activated T cells (Bechmann et al., 2002
) and apoptosis in neurons (Deshpande et al., 2005
), but the mechanism may not necessarily be applied to certain neuronal populations. Indeed, we have investigated the contribution of Fas-specific death within the TMT injury model through immunohistochemical detection of the Fas/CD95 receptor in the dentate gyrus, and its involvement appears to be minimal (data not shown). In injury models where Fas/FasL activation is a significant contributor to neuronal apoptosis, the upregulation of the intracellular phosphatase FAP may provide a mechanism for neuronal survival and contribute to the neuroprotection often seen with the absence of IL-1R1 stimulation. Finally, the possibility that distinct apoptotic mechanisms are contributing to the neuronal damage seen in each group cannot be excluded. It is possible that in the absence of IL-1R1, Fas signaling may be a significant contributor to the observed cell death, but is not as active in wildtype mice. Whether the observed changes in mRNA translate into downstream signaling of Fas/FasL would require further investigation.
Given the similar levels of damage, the TMT injury offers a model to identify differential responses in IL-1R1−/− mice that may contribute to the neuroprotection often seen in other models of brain injury. Inhibition of IL-1 activity on IL-1R1 through various interventions has been proposed as a possible therapeutic intervention for brain injury in the human population. However, data from the current study suggests that the neuroprotective effects of IL-1 inhibition may be injury specific and may not be a process that can be generalized. While IL-1β has been implicated in neurodegeneration, there are also observations indicating that, under certain conditions, IL-1 can contribute beneficial effects (Brenneman et al., 1992
), including regulation of ischemic tolerance in CA1 hippocampal neurons (Ohtsuki et al., 1996
), anti-convulsive properties such as, slowing the rate of amygdala kindling (Sayyah et al., 2005
), and the induction of various neurotrophic factors. In addition, previous evidence reveals that IL-1 demonstrates properties signifying its role as a modulator of memory functions, including effects on long term potentiation and synaptic transmission (Bellinger et al., 1993
), as well as, regulating N-methyl-D-aspartate (NMDA) receptors (Ma et al., 2002
) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Lai et al., 2006
). These properties of IL-1 suggest additional intracellular interactions that have yet to be identified. Thus, future efforts to target IL-1 signaling for therapeutic intervention require a greater level of understanding of the cell biology associated with IL-1 and its pleitrophic actions in the brain.