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. 2011 Dec 7;31(49):18048-59.
doi: 10.1523/JNEUROSCI.4067-11.2011.

Neuron-specific effects of interleukin-1β are mediated by a novel isoform of the IL-1 receptor accessory protein

Yangyang Huang  1 , Dirk E Smith, Osvaldo Ibáñez-Sandoval, John E Sims, Wilma J Friedman
Affiliations

Neuron-specific effects of interleukin-1β are mediated by a novel isoform of the IL-1 receptor accessory protein

Yangyang Huang et al. J Neurosci. .

Abstract

In the CNS, interleukin-1β (IL-1β) is synthesized and released during injury, infection, and disease, mediating inflammatory responses. However, IL-1β is also present in the brain under physiological conditions, and can influence hippocampal neuronal function. Several cell-specific IL-1-mediated signaling pathways and functions have been identified in neurons and astrocytes, but their mechanisms have not been fully defined. In astrocytes, IL-1β induced both the p38 MAPK and NF-κB (nuclear factor κB) pathways regulating inflammatory responses, however in hippocampal neurons IL-1β activated p38 but not NF-κB. Additionally, IL-1β induced Src phosphorylation at 0.01 ng/ml in hippocampal neurons, a dose 1000-fold lower than that used to stimulate inflammatory responses. IL-1 signaling requires the type 1 IL-1 receptor and the IL-1 receptor accessory protein (IL-1RAcP) as a receptor partner. We previously reported a novel isoform of the IL-1RAcP, IL-1RAcPb, found exclusively in CNS neurons. In this study, we demonstrate that AcPb specifically mediates IL-1β activation of p-Src and potentiation of NMDA-induced calcium influx in mouse hippocampal neurons in a dose-dependent manner. Mice lacking the AcPb, but retaining the AcP, isoform were deficient in IL-1β regulation of p-Src in neurons. AcPb also played a modulatory role in the activation of p38 MAPK, but had no effect on NF-κB signaling. The restricted expression of AcPb in CNS neurons, therefore, governs specific neuronal signaling and functional responses to IL-1β.

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Figures

Figure 1.
Figure 1.
Expression of IL-1R1, AcP and AcPb in primary mouse hippocampal neurons and astrocytes. A, Reverse transcriptase-PCR amplification of AcP, AcPb and IL-1RI mRNAs from hippocampal neurons and astrocytes, β actin was used as control. NTC was control without cDNA for PCR. B, Relative expression of AcP and AcPb mRNA levels in hippocampal neurons and astrocytes quantified by real-time PCR. All mRNAs are expressed relative to the neuronal expression level of AcP, which was normalized to 1, using actin as an internal control (data from three different experiments with triplicate loading each). C, IP-Western blots for AcP and AcPb in hippocampal neurons and astrocytes. Protein (50 μg) from astrocytes transfected with AcP or AcPb was used as positive control in the left two lanes. A total of 500 μg of protein from primary neurons or astrocytes was immunoprecipitated with a pan-AcP monoclonal antibody and immunoblots were analyzed with a pan-AcP antiserum.
Figure 2.
Figure 2.
AcPb does not regulate NF-κB signaling. A, IL-1β induced phosphorylation of IκB in WT and AcPb−/− astrocytes but not total AcP−/− astrocytes. Cultured hippocampal astrocytes were treated with IL-1β (10 ng/ml) for the indicated times and lysates were analyzed by Western blot for phospho-IκB, stripped and reprobed for total IκB and β actin. The blot shown is representative of three independent experiments. B, p65 NF-κB subunit translocated to the nucleus in WT and AcPb−/− astrocytes but not AcP−/− astrocytes. Astrocytes were treated with IL-1β (10 ng/ml) for 2 h, fixed with 4%PFA, and immunostained for p65 (red). Cell nuclei were labeled with DAPI (blue). C, κB-luciferase activity is induced by IL1β in WT and AcPb−/−astrocytes, but not AcP−/− astrocytes. Astrocytes were transfected with κB-driven luciferase reporter plasmids. Twenty-four hours after transfection, cells were treated with IL-1β (10 ng/ml) for 4 h. Protein lysates were analyzed for luciferase activity. Data are from four different experiments with triplicate loading each. Student's t test was used to evaluate data from cells treated with or without IL-1β. *p < 0.01. D, IL-6 mRNA was induced by IL1β (10 ng/ml) in WT and AcPb−/− astrocytes but not total AcP−/− astrocytes by real-time PCR analysis. Graph depicts 6 h IL-1β treatment. mRNA levels were normalized to wild-type control. E, No nuclear translocation of p65 was induced by 2 h IL-1β (10 ng/ml) in hippocampal neurons. F, No phosphorylation of IκB was detected in WT, total AcP−/− or AcPb−/− neurons in response to IL-1β (10 ng/ml). p-IκB in astrocytes was used as positive control. The blot shown is representative of three independent experiments.
Figure 3.
Figure 3.
Transfection of AcPb into WT astrocytes does not inhibit IκB phosphorylation induced by IL-1β. WT astrocytes were transfected with AcPb or an empty vector. Forty-eight hours after transfection, cells were treated with IL-1β (10 ng/ml) for 5 min. Protein lysates were immunoblotted for p-IκB and AcPb, then stripped and reprobed for IκB and actin as control. The blot shown is representative of four independent experiments.
Figure 4.
Figure 4.
IL-1β induced p38 phosphorylation in hippocampal astrocytes and neurons. A, Time course of IL-1β (10 ng/ml)-induced p38 phosphorylation in WT, total AcP−/−, and AcPb−/− astrocytes. B, Quantification of blots from four different experiments as in A. Densitometric values were expressed as p-p38/total p38, and then normalized to the treatment at 10 min (peak value). P-p38 was not detected in control samples, therefore the values were set at 0. C, Time course of IL-1β (10 ng/ml)-induced p38 phosphorylation in WT, total AcP−/−, and AcPb−/− neurons. D, Quantification of blots from four different experiments as in C. Densitometric values were expressed as p-p38/total p38, and then normalized to untreated cells (0 min) for each genotype. E, Dose–response of IL-1β-induced p38 phosphorylation after 20 min in WT, total AcP−/−, and AcPb−/− neurons. F, Quantification of blots from three different experiments as in E. Densitometric values were expressed as p-p38/total p38, and then normalized to untreated cells (0 ng/ml IL-1) for each genotype. The significance was determined by one-way ANOVA with Tukey's post hoc analysis. The p value equals to 0.0023 between WT and AcPb−/− neurons treated with 100 ng/ml IL-1. Asterisks indicate values significantly different from control at *p < 0.05, **p < 0.001, and ***p < 0.0001.
Figure 5.
Figure 5.
Src phosphorylation by IL-1β (0.01 ng/ml) in hippocampal neurons is dependent on AcPb. A, Dose–response treatment of IL-1β applied to cultured WT mouse hippocampal neurons for 20 min. Protein lysates were immunoblotted for p-Src and reprobed for total Src. Quantification of blots from three different experiments is shown below the blot. Densitometric values were expressed as p-Src/total Src, and then normalized to untreated cells (0 ng/ml). B, Time course of Src phosphorylation induced by IL-1β in mouse hippocampal neurons. At low concentration (0.01 ng/ml), Src is phosphorylated by IL-1β in WT neurons, but not in total AcP−/− or AcPb−/− neurons. Quantification of blots from three different experiments is shown below the blot. The significance was determined by one-way ANOVA with Tukey's post hoc analysis. Asterisks indicate values significantly different from control at *p < 0.05, ***p < 0.0001.
Figure 6.
Figure 6.
Effects of overexpressing AcPb in astrocytes on IL-1β-induced p-Src and p-p38 signaling. A, Time course of IL-1β (0.01 ng/ml) treatment of WT astrocytes with and without AcPb transfection. Expression of AcPb in WT astrocytes enabled IL-1β to induce Src phosphorylation. B, IL-1 (0.01 ng/ml)-induced p-Src in total AcP−/− astrocytes restored with AcPb but not AcP. In contrast, restoration of AcP, but not AcPb was sufficient to restore IL-1β induction of p-p38 MAPK, even at the low dose. C and D show quantification of p-Src and p-p38 MAPK, respectively, from blots from three different experiments. Densitometric values were expressed as p-Src/total Src (C) or p-p38/total p38 (D), and then normalized to untreated cells (0 min). The significance was determined by one-way ANOVA with Tukey's post hoc analysis. Asterisk indicates values significantly different from control at p < 0.05. E, High dose of IL-1 (10 ng/ml)-induced p-p38 in total AcP−/− astrocytes restored with either AcP or AcPb, but only induced p-IκB in total AcP−/− astrocytes restored with AcP. F, Quantification of p-p38 induction in the tAcP−/− astrocytes transfected either with AcP or AcPb and treated with the high dose (10 ng/ml) of IL-1 for 5 min. Note in B and E that although equal amounts of plasmid DNA for AcP or AcPb were used for transfections, expression of AcPb is always lower than AcP. Thus, when normalized to the amount of AcP or AcPb expressed, the level of p-p38 activation was similar.
Figure 7.
Figure 7.
IL-1β (0.01 ng/ml) induced transient phosphorylation of NR2B within 5 min in WT neurons. Graph shows quantification of two independent experiments.
Figure 8.
Figure 8.
AcPb is required for IL-1β (0.01 ng/ml)-potentiated calcium influx induced by NMDA in hippocampal neurons. A–C, Representative changes in [Ca2+]i measured by Fura-2 in response to IL-1β and NMDA in WT, total AcP−/− and AcPb−/− neurons. Data are shown as the ratio of 340/380 fluorescence emission. A, WT neurons were pretreated with or without 0.01 ng/ml IL-1β, PP2 + 0.01 ng/ml IL-1β, or 10 ng/ml IL-1β before the addition of NMDA. B, C, AcPb−/− neurons (B) or total AcP−/− neurons (C) were pretreated with or without IL-1β (0.01 ng/ml) before NMDA addition. Baseline recordings were established for 4 min followed by treatment with or without IL-1β for 6 min. NMDA (10 μm) was applied for 7 min and then washed away. At the end of each recording, KCl (30 mm) was applied to ensure calcium response and cell viability. D, Quantification of NMDA-induced calcium influx from 3 to 5 individual cultures. Area under the curve (AUC) from each group was normalized to WT neuron control, which was set at 100%. Total number of neurons analyzed for a given group is as follows: WT neuron, control (CTR), 300; WT neuron, 0.01 ng/ml IL-1β, 377; WT neuron, PP2 + 0.01 ng/ml IL-1β, 125; WT neuron, 10 ng/ml IL-1β, 73; total AcP−/− neuron, CTR, 155; total AcP−/− neuron, 0.01 ng/ml IL-1β, 158; AcPb−/− neuron, CTR, 315; AcPb−/− neuron, 0.01 ng/ml IL-1β, 271.
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References

    1. Andre R, Lerouet D, Kimber I, Pinteaux E, Rothwell NJ. Regulation of expression of the novel IL-1 receptor family members in the mouse brain. J Neurochem. 2005;95:324–330. - PubMed
    1. Avital A, Goshen I, Kamsler A, Segal M, Iverfeldt K, Richter-Levin G, Yirmiya R. Impaired interleukin-1 signaling is associated with deficits in hippocampal memory processes and neural plasticity. Hippocampus. 2003;13:826–834. - PubMed
    1. Balosso S, Maroso M, Sanchez-Alavez M, Ravizza T, Frasca A, Bartfai T, Vezzani A. A novel non-transcriptional pathway mediates the proconvulsive effects of interleukin-1beta. Brain. 2008;131:3256–3265. - PMC - PubMed
    1. Bellinger FP, Madamba S, Siggins GR. Interleukin 1β inhibits synaptic strength and long-term potentiation in the rat CA1 hippocampus. Brain Res. 1993;628:227–234. - PubMed
    1. Berglöf E, Andre R, Renshaw BR, Allan SM, Lawrence CB, Rothwell NJ, Pinteaux E. IL-1Rrp2 expression and IL-1F9 (IL-1H1) actions in brain cells. J Neuroimmunol. 2003;139:36–43. - PubMed

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