The synergistic interactions between TNF-α and AVP in the choroid plexus epithelium as assessed by the level of activation of JNK and its target transcription factors c-Jun and ATF2.

<p>The activation of the JNK signaling cascade in the Z310 cell line was assessed after 1-h incubation with either TNF-α (5 ng/ml) or a combination of TNF-α (5 ng/ml) and AVP (1 nM). In non-radioactive JNK activity assays, 200 ng of recombinant (rec) human c-Jun protein was used as a substrate for JNK. The extent of activation of c-Jun and ATF2 was assessed by Western blotting (30 µg of total protein per lane was loaded). The representative immunoblots are shown based on three independent experiments. The ratios of optical density of bands for phosphorylated proteins over the optical density of bands for the total (phosphorylated and non-phosphorylated) proteins are shown.</p

Post-traumatic activation of JNK in the lateral ventricle choroid plexus (CP) as assessed by Western blotting and JNK activity assays at 6 h after injury.

<p>A comparison of AVP-deficient Brattleboro rats (<i>Avp</i>^di/di) with their parental Long-Evans strain (WT). The activity of JNK was assessed in both the ipsilateral (Ipsi CP/I) and contralateral (Contr CP/C) CPs. For Western blotting, 40 µg of total protein per lane was loaded. In non-radioactive JNK activity assays, 200 ng of recombinant (rec) human c-Jun protein was used as a substrate for JNK. Similar results were obtained in an independent experiment involving separate groups of animals. The ratios of optical density of bands for phosphorylated proteins over the optical density of bands for the total (phosphorylated and non-phosphorylated) proteins are shown (<i>n</i> = 4 per group).</p

Signal transduction pathways involved in the synergistic interactions between TNF-α and AVP.

<p>The experiments were conducted on the choroid plexus epithelial cell line Z310 and changes in production of CXCL1 were assessed by real-time RT-PCR. To inhibit JNK (SP600125; 10 µM) or p38 (SB203580; 1 µM) activity, or to interfere with ERK (MEK1 inhibitor PD-98059; 10 µM) or NF-κB (SN50; 100 µg/ml) signaling, the Z3110 cells were pre-incubated for 1 h with respective inhibitors. These inhibitors were also added to the culture media when the cells were exposed to TNF-α (5 ng/ml) or a combination of TNF-α (5 ng/ml) and AVP (1 nM) for an additional hour. Note that the selective JNK inhibitor SP600125 completely abolished the induction of CXCL1 synthesis observed in response to TNF-α alone or to a combination of TNF-α and AVP. A selective MEK1 inhibitor PD-98059 partially (by >50%) inhibited CXCL1 synthesis, whereas p38 inhibitor SB203580 had no effect. In comparison, the inhibition of NF-κB signaling resulted in the amplification of chemokine synthesis. *<i>p</i><0.05, **<i>p</i><0.01 for treatment versus control. ^†<i>p</i><0.05, ^††<i>p</i><0.01 for the incubation with inhibitor versus the incubation without inhibitor (<i>n</i> = 3 per group).</p

The synergistic interactions between TNF-α and AVP (A), and IL-1β and AVP (B) in the choroid plexus epithelium as assessed by real-time RT-PCR.

<p>Changes in production of CXCL1 in the choroid plexus epithelial cell line Z310 were assessed after 1-h incubation with either the cytokine (TNF-α or IL-1β) alone or a combination of the cytokine (TNF-α or IL-1β) and AVP. The exposure of Z310 cells to AVP alone at a concentration ranging between 0.1 nM and 1 µM resulted in only small increases in CXCL1 synthesis (a maximum increase observed was 6-fold relative to control with AVP at 100 nM). *<i>p</i><0.05, **<i>p</i><0.01 for treatment versus control. ^†<i>p</i><0.05, ^††<i>p</i><0.01 for TNF-α + AVP versus TNF-α alone or for IL-1β + AVP versus IL-1β alone (<i>n</i> = 3–4 per group).</p

The effect of inhibition of JNK in vivo.

<p>To inhibit the activity of JNK in rats sustaining TBI, a selective JNK inhibitor SP600125 (JNK Inh) was injected i.p. at 4 and 8 h post-TBI at a dose of 30 mg/kg for each injection. A control group of rats was injected with vehicle (Veh; DMSO). (A) The efficacy of pharmacological inhibition of JNK as assessed by the reduction in post-traumatic activation of ATF2 in the lateral ventricle choroid plexus (CP) at 6 h after injury. Another target transcription factor for JNK, c-Jun, was undetectable in the CP. The activity of ATF2 was assessed by Western blotting in both the ipsilateral (Ipsi CP/I) and contralateral (Contr CP/C) CPs (30 µg of total protein per lane was loaded). The representative immunoblots are shown based on two independent experiments involving separate groups of animals. The ratios of optical density of bands for phosphorylated ATF2 over the optical density of bands for the total (phosphorylated and non-phosphorylated) ATF2 are shown. (B) The effect of JNK inhibition on post-traumatic production of neutrophil chemoattractants CXCL1–3 by the lateral ventricle CP as assessed by real-time RT-PCR at 6 h after injury. Cyclophilin A (Cycl-A) was used for the normalization of the data. (C) The role of AVP in promoting the post-traumatic influx of neutrophils across the blood-cerebrospinal fluid barrier (BCSFB) and the effect of JNK inhibition on this influx. To define the pathophysiological role of AVP in promoting neutrophil influx across the BCSFB, AVP-deficient Brattleboro rats (<i>Avp</i>^di/di) were compared with their parental Long-Evans strain (WT). The magnitude of neutrophil influx was assessed by the myeloperoxidase (MPO) activity assays at 24 h post-TBI. *<i>p</i><0.05 for JNK Inh versus Veh or for <i>Avp</i>^di/di versus WT (<i>n</i> = 4–6 per group).</p

The synergistic interactions between TNF-α and AVP in the choroid plexus epithelium as assessed by real-time RT-PCR (A) and ELISA (B).

<p>The choroid plexus epithelial cells Z310 were incubated for the indicated periods of time with either TNF-α alone (5 ng/ml) or a combination of TNF-α (5 ng/ml) and AVP (1 nM). Cyclophilin A (Cycl-A) was used for the normalization of RT-PCR data. The concentrations of proinflammatory mediators were measured in the cell culture media collected at the end of the experiment. *<i>p</i><0.05, **<i>p</i><0.01 for treatment versus control. ^†<i>p</i><0.05, ^††<i>p</i><0.01 for TNF-α + AVP versus TNF-α alone (<i>n</i> = 3–4 per group).</p

Post-traumatic synthesis of proinflammatory mediators by the lateral ventricle choroid plexus (CP) as assessed by real-time RT-PCR.

<p>A comparison of AVP-deficient Brattleboro rats (<i>Avp</i>^di/di) with their parental Long-Evans strain (WT). The production of proinflammatory mediators in the ipsilateral (Ipsi CP) and contralateral (Contr CP) CPs is shown. Cyclophilin A (Cycl-A) was used for the normalization of the data. *<i>p</i><0.05, **<i>p</i><0.01 for the ipsilateral versus contralateral CP. ^†<i>p</i><0.05, ^††<i>p</i><0.01 for <i>Avp</i>^di/di versus WT rats (<i>n</i> = 6–9 per rat strain per time point).</p