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. 2006 Feb 21;103(8):2904-8.
doi: 10.1073/pnas.0510960103. Epub 2006 Feb 13.

Ceramide mediates the rapid phase of febrile response to IL-1beta

Affiliations

Ceramide mediates the rapid phase of febrile response to IL-1beta

Manuel Sanchez-Alavez et al. Proc Natl Acad Sci U S A. .

Abstract

IL-1beta was identified after a long search for the endogenous pyrogen. It acts by inducing synthesis of prostaglandin E2, which mediates the late phase of IL-1beta-induced fever. Here we show by radiotelemetry that the early phase of the fever response to IL-1beta is mediated by ceramide. Hypothalamic application of the cell-penetrating C2-ceramide mimics the rapid phase of the IL-1beta-induced fever. Inhibition of ceramide synthesis blocks the rapid phase of fever but does not affect the slower prostaglandin E2-dependent phase, which is blocked by indomethacin or by null mutation of the EP3 prostanoid receptor. Electrophysiological experiments on preoptic area/anterior hypothalamic neurons show that C2-ceramide, but not dihydroceramide, mimics the rapid hyperpolarizing effects of IL-1beta on the activity of warm-sensitive hypothalamic neurons. IL-1beta-mediated hyperpolarization is blocked by PP2, the selective inhibitor of the protein tyrosine kinase Src, which is known to be activated by ceramide. These in vivo and in vitro data suggest that ceramide fulfills the criteria for an endogenous pyrogen.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Dose–response curve for IL-1β and C2-ceramide on CBT after microinjection in the AH. (A) Dose–response curve for IL-1β-induced fever. CBT increased significantly at 60 min in IL-1β treated at 1, 5 and 15 μg/kg compared with IL-1β 15 ng/kg and 150 ng/kg or vehicle (∗, P < 0.05; data are averaged at 20-min intervals). (B) Dose–response curve for C2-ceramide-induced fever. CBT increased significantly at 40 min in C2-ceramide treated at doses 10, 20, 40, and 80 μg/kg (∗, P < 0.05, compared with vehicle). (C) Simultaneous recording of temperature in the POA and CBT comparing C2-ceramide 40 μg/kg, IL-1β 15 μg/kg, and vehicle control. C2-ceramide-induced rise in CBT peaks at 40 min, whereas the maximum rise in CBT caused by IL-1β peaks at 60 min. The effect of C2-ceramide declines after 60 min, reaching the baseline at ≈2 h, whereas the IL-1β-induced rise in CBT at 60 min remains at a constant level for the following 3 h, after which it declines but is significantly above the baseline (8–12 h) after injection. (A–C) All data points are from six animals each. (D) Location of cannulae aiming the AH and track of the thermisor to the POA; see arrows. Slices correspond to anterior–posterior coordinates from Bregma, −0.46 for AH and 0.38 for POA.
Fig. 2.
Fig. 2.
Inhibition of the different phases of fever response by intrahypothalamic application of N-Smase inhibitor, spiroepoxide, or i.p.-administered indomethacin. Pretreatment (pt) was followed by second treatment (2nd) (see arrows). (A) N-Smase inhibitor 10 μM pretreatment inhibited partially the effect of IL-1β at 40 and 60 min; F(3, 16) = 13.887; ∗, P < 0.01, vehicle + IL-1β vs. vehicle + vehicle, N-Smase inhibitor 10 μM + IL-1β 15 μg/kg, and N-Smase inhibitor 10 μM + vehicle. After 60 min, the vehicle + IL-1β and N-Smase inhibitor 10 μM + IL-1β 15 μg/kg differ with vehicle + vehicle and N-Smase inhibitor 10 μM + vehicle groups; F(3, 16) = 38.741, P < 0.01. (B) Pretreatment with indomethacin does not have an effect on the early phase of the IL-1β increase on CBT at 40 min (see +, F(3, 16) = 18.541, P < 0.05, vehicle + IL-1β vs. indomethacin + vehicle, vehicle + vehicle) but interferes with the development of the fever response after 60 min (F(3, 16) = 21.342; ▾, P < 0.01). (C) The rapid rise in CBT in response to IL-1β is unaffected by the null mutation of the prostanoid receptor type 3; EP3R (paired t test, +, P < 0.05 at 60 min and ∗, P < 0.01 at 80 min, vehicle vs. IL-1β 150 ng/kg).
Fig. 3.
Fig. 3.
IL-1β and C2-ceramide decrease the firing rate and reduce the thermosensitivity of warm-sensitive POA/AH neurons. (A) Current-clamp recordings showing the response of a warm-sensitive neuron to a temperature increase from 37 to 40.3°C before (Upper) and after application of 0.3 nM IL-1β (Lower). IL-1β hyperpolarized the neuron by ≈6 mV and reduced its thermosensitivity from 0.9 spikes per s−1°C−1 to 0.1 spikes per s−1°C−1. (B) Current-clamp recordings showing the response of a warm-sensitive neuron to a temperature increase from 36.8 to 39.7°C before (Upper) and after (Lower) application of 10 μM C2-ceramide. C2-ceramide hyperpolarized the neuron by ≈4 mV and reduced its thermosensitivity from 1.1 spikes per s−1°C−1 to 0.2 spikes per s−1°C−1. (C) Time course of the IL-1β (0.3 nM) hyperpolarization. (D) Preincubation of the neurons with the selective Src inhibitor PP2 (10 μM) for 20 min prevents the effect of IL-1β.
Fig. 4.
Fig. 4.
Schematic model of the fast and slow phases of IL-1β-induced fever. The fast response (0–30 min) to IL-1β to a large extent is mediated by ceramide. The covalent modification of ion channels by ceramide-activated Src could be responsible for the fast neuronal effect of the cytokine. The slow phase of the IL-1β-induced fever depends on NF-κB-mediated transcription of COX2 and production PGE2 that activates prostanoid receptors on neurons. Indomethacin, a COX1/2 inhibitor, blocks PGE2 synthesis; spiroepoxide, an N-Smase inhibitor, inhibits the production of ceramide; and PP2, a selective inhibitor of Src, inhibits protein tyrosine phosphorylation by Src.

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