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Comparative Study
. 2005 Feb 1;102(5):1755-60.
doi: 10.1073/pnas.0409225102. Epub 2005 Jan 21.

A cascade of cytokines mediates mechanical inflammatory hypernociception in mice

T M Cunha  1 , W A Verri Jr, J S Silva, S Poole, F Q Cunha, S H Ferreira
Affiliations
Comparative Study

A cascade of cytokines mediates mechanical inflammatory hypernociception in mice

T M Cunha et al. Proc Natl Acad Sci U S A. .

Abstract

The hypernociceptive effects of cytokines [TNF-alpha, keratinocyte-derived chemokine (KC), and IL-1beta] and their participation in carrageenan (Cg)-induced inflammatory hypernociception in mice were investigated. Nociceptor sensitization (hypernociception) was quantified with an electronic version of the von Frey filament test in WT and TNF receptor type 1 knockout mice (TNF-R1-/-). TNF-alpha-induced hypernociception was abolished in TNF-R1-/- mice, partially inhibited by pretreatment with IL-1 receptor antagonist (IL-1ra) or indomethacin and unaffected by Ab against KC (AbKC) or guanethidine. IL-1ra and indomethacin pretreatment strongly inhibited the hypernociception induced by IL-1beta, which was not altered by AbKC or guanethidine or by knocking out TNF-R1. KC-induced hypernociception was abolished by AbKC, inhibited by pretreatment with indomethacin plus guanethidine, and partially inhibited by IL-1ra, indomethacin, or guanethidine. In contrast, KC-induced hypernociception was not altered by knocking out TNF-R1. Cg-induced hypernociception was abolished by administration of indomethacin plus guanethidine, diminished in TNF-R1-/- mice, and partially inhibited in WT mice pretreated with AbKC, IL-1ra, indomethacin, or guanethidine. TNF-alpha, KC, and IL-1beta concentrations were elevated in the skin of Cg-injected paws. The TNF-alpha and KC concentrations rose concomitantly and peaked before that of IL-1beta. In mice, the cytokine cascade begins with the release of TNF-alpha (acting on TNF-R1 receptor) and KC, which stimulate the release of IL-1beta. As in rats, the final mediators of this cascade were prostaglandins released by IL-1beta and sympathetic amines released by KC. These results extend to mice the concept that the release of primary mediators responsible for hypernociception is preceded by a cascade of cytokines.

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Figures

Fig. 1.
Fig. 1.
Mechanical hypernociception induced by i.pl. injection of IL-1β: the role of TNF-α, KC, prostanoides, and sympathomimetic amines. (A) Dose– and time–response curves of the hypernociception induced by i.pl. injection of IL-1β (10, 100, and 1,000 pg per paw) or saline (SAL; control). The hypernociceptive effects were determined at 1, 3, and 5 h after the stimuli injection. (B) Nociceptive response induced by IL-1β (1,000 pg per paw) in WT mice pretreated with 15 μl of saline (Sal) or TNF-R1–/– mice and pretreatment of WT mice with 500 ng of AbKC, 500 ng of IL-1ra, 5 mg/kg indomethacin (Indo) delivered i.p., 30 mg/kg guanethidine (Gua) delivered s.c., and indomethacin plus guanethidine. The hypernociceptive responses were taken 3 h after IL-1β injection. The results are expressed by the mean ± SEM of five animals per group. *, Statistically significant different time points (P < 0.05); **, statistically significant difference compared with the control group (P < 0.05).
Fig. 2.
Fig. 2.
Mechanical hypernociception induced by i.pl. injection of KC: the role of TNF-α, IL-1β, prostaglandins, and sympathomimetic amines. (A) Dose– and time–response curves of the hypernociception induced by i.pl. injection of KC (1, 3, and 10 pg per paw) or saline (SAL; control). The hypernociceptive effects were determined at 1, 3, and 5 h after the stimuli injection. (B) Nociceptive response induced by KC (10 ng per paw) in WT mice pretreated with 15 μlof saline (Sal) or TNF-R1–/– mice and pretreatment of WT mice with 500 ng of AbKC delivered i.pl., 500 ng of IL-1ra delivered i.pl, 5 mg/kg indomethacin (Indo) delivered i.p., 30 mg/kg guanethidine (Gua) delivered s.c., and indomethacin plus guanethidine. The hypernociceptive responses were taken 3 h after KC injection. The results are expressed as the mean ± SEM of five animals per group. *, Statistically significant different time points (P < 0.05); **, statistically significant difference compared with the control group (P < 0.05).
Fig. 6.
Fig. 6.
Production of cytokines in TNF-R1–/– mice. Concentration of KC (A) and IL-1β (B) in WT and TNF-R1–/– mice paws injected with 100 μg of Cg, 100 pg of TNF-α, 10 ng of KC, or 25 μl of saline (Sal) or in naïve paws. At 3 h after injection, mice were killed, and paw skin samples were extracted for cytokines, which were measured by ELISA. The results are expressed by the mean ± SEM of four animals per group. *, Statistically significant differences compared with the saline group (P < 0.05); **, statistically significant differences compared with the WT mice group (P < 0.05).
Fig. 3.
Fig. 3.
Mechanical hypernociception induced by i.pl. injection of TNF-α: the role of TNF-R1 receptor, KC, IL-1β, prostaglandins, and sympathomimetic amines. (A) Dose– and time–response curves of the hypernociception induced by i.pl. injection of TNF-α (1, 10, 100, and 1,000 pg per paw) or saline (SAL; control). The hypernociceptive effects were determined at 1, 3, and 5 h after the stimuli injection. (B) Nociceptive response induced by TNF-α (100 pg per paw) in WT mice treated with 20 μl of saline (Sal) or TNF-R1–/– mice and pretreatment of WT mice with 500 ng of AbKC delivered i.pl. 500 ng of IL-1ra delivered i.pl. 5 mg/kg indomethacin (Indo) delivered i.p., 30 mg/kg guanethidine (Gua) delivered s.c., and indomethacin plus guanethidine. The hypernociceptive responses were taken 3 h after TNF-α injection. The results are expressed as the mean ± SEM of five animals per group. *, Statistically significant different time points (P < 0.05); **, statistically significant difference compared with the control group (P < 0.05).
Fig. 4.
Fig. 4.
Mechanical hypernociception induced by i.pl. injection of Cg in WT and TNF-R1–/– mice: effects of IL-1ra, AbKC, indomethacin, guanethidine, and association of indomethacin and guanethidine. (A) Dose– and time–response curves of the hypernociception induced by i.pl. injection of Cg (30, 100, and 300 μg per paw) or saline (SAL; control). The hypernociceptive effects were determined at 1, 3, and 5 h after the stimuli injection. (B) Nociceptive response induced by Cg (100 μg per paw) in WT mice pretreated with 15 μl of saline (Sal) or TNF-R1–/– mice and pretreatment of WT mice with 500 ng of AbKC delivered i.pl., 500 ng of IL-1ra delivered i.pl., 5 mg/kg indomethacin (Indo) delivered i.p., 30 mg/kg guanethidine (Gua) delivered s.c., and indomethacin plus guanethidine. The hypernociceptive responses were made 3 h after Cg injection. The results are expressed by the mean ± SEM of five animals per group. *, Statistically significant different time points (P < 0.05); **, statistically significant difference compared with the control group (P < 0.05).
Fig. 5.
Fig. 5.
I.pl. injection of Cg-stimulated TNF-α, KC, and IL-1β production in paw skin. Concentrations of TNF-α (A), KC (B), and IL-1β (C) in mice paws injected with 100 μg of Cg or saline (Sal) or in naive paws. At 0.5, 1, 3, and 5 h after injection, mice were killed and paw skin samples were extracted for cytokines, which were measured by ELISA. The results are expressed by the mean ± SEM of four animals per group. *, Statistically significant differences compared with the control group (P < 0.05).
Fig. 7.
Fig. 7.
Representative diagram of Cg-induced inflammatory hypernociceptive cytokine cascade in mice. Shown is the sequential release (solid line, stimulation) of inflammatory mediators initiated by Cg and intermediated by cytokines and the final induction by prostanoids (PGs) and sympathetic amines (SA). The cascade pharmacological and genetic approach susceptibility is also shown (dotted line, inhibition) along with the other possibilities for TNF-α action (question marks).
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