Research ReportThe type 1 interleukin 1 receptor is not required for the death of murine hippocampal dentate granule cells and microglia activation
Introduction
Neuronal death following injury activates many cellular changes in the damaged tissue area. In addition to the activation of local glia and often the recruitment of blood-borne leukocytes, local synthesis of inflammation-related cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα), exerts a wide range of effects. These effects are mediated primarily by IL-1 type 1 receptor (IL-1R1) and TNF receptor 1 (TNFp55R), respectively. While a pathophysiological role for IL-1 as part of the inflammatory process in the brain has been established (Rothwell and Luheshi, 2000, Allan and Rothwell, 2001), exactly how the signaling process may contribute to neuronal death and protection is still unclear. However, data clearly support its role as a key mediator of inflammation and neuronal death in acute brain injuries, such as stroke and trauma (Allan et al., 2005).
IL-1α and IL-1β are the more characterized forms of IL-1 (Rothwell and Luheshi, 2000). An increase in IL-1β expression has been demonstrated under various neurodegenerative conditions, as well as experimental models of brain injury (Griffin et al., 1989, Mogi et al., 1994, Zhao and Schwartz, 1998, Rothwell, 2003). The earliest source of IL-1 after injury appears to be microglia, possibly due to the expression of the converting enzyme caspase 1 (Eriksson et al., 1999). This would allow for a rapid production of the active form of IL-1β upon cleavage of pro-IL-1β. Upon stimulation, IL-1 can induce release of growth factors, decrease glutamate release, enhance the effects of GABA, increase the activation of inducible nitric oxide synthase, and increase the production of inflammatory mediators, thus establishing a cycle of inflammation (Rothwell and Luheshi, 2000, Boutin et al., 2001, Griffin, 2006).
Many of the detrimental effects of IL-1 are thought to occur through the activation of IL-1R1 (Dinarello, 1996). IL-1R1 binding initiates a sequence of protein–protein interactions forming a complex to recruit the IL-1 receptor accessory protein (IL-1RAcP) and induce cell signaling (Greenfeder et al., 1995). Subsequent binding of the IL-1β/IL-1R1 complex with the adaptor protein, myeloid differentiation (MyD) 88, facilitates the incorporation and phosphorylation of interleukin-1 receptor-associated kinase IRAK (Wesche et al., 1997). The phosphorylation of IRAK can lead to the activation of the mitogen-activated protein kinase, c-Jun N-terminal kinase, JNK (O'Neill and Green, 1998). The induction of JNK via IL-1β signaling has been reported in various cell types and within the hippocampus (Uciechowski et al., 1996, Vereker et al., 2000).
The involvement of IL-1 in excitotoxicity, seizure activity, memory, and depression suggests a susceptibility to IL-1 effects in the hippocampus. This has been supported by the differential effect of IL-1β on excitotoxicity, with the increased death of primary hippocampal neurons (Viviani et al., 2003), yet a reduction in death of cultured cortical neurons (Strijbos and Rothwell, 1995). Granule cells of the dentate gyrus and CA1 and CA3 pyramidal neurons express IL-1R1 (Cunningham et al., 1992, Ericsson et al., 1995). In addition, neutralization of IL-1 actions on IL-1R1 by the IL-1 receptor antagonist (IL-1ra) offers protection to neurons against ischemia, trauma, excitotoxic damages, and chemical-induced seizures (Touzani et al., 1999, Toulmond and Rothwell, 1995). A transient decrease in receptor densities in the dentate gyrus of the hippocampus has been reported within 24 h following focal trauma, coinciding with an increase in IL-1ra (Gabellec et al., 1999). In the cortex, the role for IL-1R1 has been characterized and demonstrated to be associated with the progressive neuronal degeneration that follows a hypoxic–ischemic insult (Basu et al., 2002, Basu et al., 2005). Basu et al. (2005) found that while mice lacking IL-1R1 showed a limited protection from an the acute injury response, they did show neuroprotection with regards to the second wave of neuronal death and the progressive enlargement of infarct area suggested to occur as a result of neuroinflammation.
Dentate granule neurons are not often considered to be a vulnerable population; however, damage to this cell population has been reported in experimental models of ischemia, hypoxia, and seizures (Harry and Lefebvre d'Hellencourt, 2003 review). While IL-1 receptor signaling has been implicated in cortical and hippocampal pyramidal cell death following trauma, ischemia, and excitotoxicity, the generalized nature of these effects and influence on the dentate granule cell population has not been examined. To investigate whether IL-1 signaling through IL-1R1 mediates damage to the dentate granule neurons, we employed a chemical injury that, in mice, initiates neuronal death within 24 h. Previous reports using the trimethyltin (TMT) murine model of brain damage indicated an associated neuroinflammatory response, including an increase in IL-1 (Bruccoleri et al., 1998, Fiedorowicz et al., 2001) and the induction of a microglia response during an active phase of neuronal death. In the current study, we evaluated the hippocampus at a time point representative of the initial stage of neuronal damage induced by TMT. Wildtype mice and mice deficient in IL-1R1 (IL-1R1−/−) were examined for changes in the severity of neuronal damage and the associated glia response as a framework to identify IL-1 involvement in the initiation of cell death within the dentate granule region. We report that the absence of IL-1R1 did not provide neuroprotection nor alter the associated microglia response. Consistent with the literature, IL-1R1−/− mice displayed an attenuated GFAP astrocyte response and we now report a significant induction of components of the Fas signaling pathway in receptor-null mice that may serve as an alternative death initiating pathway in the absence of IL-1R1 signaling.
Section snippets
Characterization of the early hippocampal histopathology in wildtype (WT) mice
In Fig. 1, representative images demonstrate immunostaining for GFAP, IB4, and active caspase 3 (AC3) in the suprapyramidal blade of the dentate gyrus in control and TMT-dosed mice. Compared to control tissue, AC3+ cells (arrow) and IB4+ microglia (arrow head) were present at 6 h post-TMT and were limited to the area along the inner blade of the dentate gyrus. By 12 h post-TMT, cell death progressed into the blades of the dentate (AC3+ cells indicated by arrow) with limited staining for
Discussion
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 in 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. This suggests
Murine model of dentate granule cell damage
Pathogen free, male weanling (25–30 days of age) C57BL/6J mice (Charles River; Raleigh, NC) were randomly assigned to experimental groups and administered a single intraperitoneal (i.p.) injection of 2.0 mg/kg trimethyltin hydroxide (TMT, originally obtained from Alfa Products, Danvers, MA) or saline, in a dosing volume of 2 ml/kg body weight. Tremor was evident at 24 h with no acute morbidity or death. Animals were individually housed in a dual corridor, semi-barrier animal facility (21 + 2 °C;
Acknowledgments
This work was supported by the division of intramural research of the National Institute of Environmental Health Science, National Institutes of Health, Department of Health and Human Services. The authors wish to thank Sue Edelstein, Dr Michelle Block and Dr. Ramen Saha for their review of the manuscript.
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