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Institut fuer Physiologie und Experimentelle Pathophysiologie, Friedrich-Alexander Universitaet, 91054 Erlangen, Germany; and
* Institut fuer Anatomie und Zellbiologie, Justus-Liebig Universitaet, Giessen, Germany
1Correspondence: Institut fuer Physiologie und Experimentelle Pathophysiologie, Universitaetsstr. 17, D-91054 Erlangen, Germany. E-mail: kress{at}physiologie1.uni-erlangen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: hyperalgesia • inflammation • cytokines • nociceptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Inflammatory hyperalgesia was prevented by experimental administration of endogenous IL-1 receptor antagonist (IL-1ra), and neutralizing antibodies to IL-1 receptors reduced pain-associated behavior in mice with experimental neuropathy (3
, 4)
. Therefore, IL-1ß is generally accepted to play a central role in the generation of thermal hyperalgesia (5)
. However, the mechanisms underlying IL-1ß contribution as well as the site of IL-1 action are controversially discussed. Increased levels of IL-1 in periphery may up-regulate cyclooxygenase type 2 production, which is associated with a centrally mediated pain hypersensitivity (6)
. However, there are studies demonstrating that IL-1ß can have antinociceptive, probably opioid-mediated effects, when administered intravenously or intraspinally (8
, 9)
. Acute peripheral nerve injury yielded glial activation and up-regulation of spinal IL-1 expression with a possible involvement in the development of behavioral hyperalgesia (7)
. Other reports favor a peripheral pronociceptive IL-1 action mediated by a complex signaling cascade and a contribution of nitric oxide, bradykinin or prostaglandins (5
, 10
11
12)
. IL-1ß sensitized abdominal visceral afferents to ischemia and histamine and excited nociceptive fibers in vivo (13
, 14)
. The first hint toward a more direct action of IL-1ß on nociceptors came from a skin nerve in vitro preparation where brief exposure to IL-1ß resulted in a facilitation of heat-evoked release of calcitonin gene-related peptide from peptidergic neurons (15)
. Due to the absence of the soma and the short latency of the effect in that study, the acute heat sensitization cannot be explained by gene expression and/or receptor up-regulation. Under pathological conditions, IL-1ß can be produced by many cell types: mononuclear cells, fibroblasts, synoviocytes, and endothelia on tissue damage (2)
. However, major cellular sources for IL-1ß in the vicinity of nociceptive nerve terminals also include glial cells and both sympathetic and sensory neurons themselves (16
, 17)
. In addition, expression of IL-1 receptor type I (IL-1RI) mRNA was detected in dorsal root ganglia neurons, suggesting a possible autocrine or paracrine influence of IL-1ß on sensory processing (17)
. The present study investigates the effects of IL-1ß on nociceptive-specific function. A potentiation of heat-activated excitatory inward currents (Iheat) was induced in isolated neurons after brief exposure to IL-1ß. RT-PCR and in situ hybridization revealed IL-1RI but not IL-1 receptor type II (IL-1RII) expression in sensory neurons. Pharmacological tools were used to address the contribution of protein tyrosine kinases (PTK) as well as protein kinase C (PKC) in the signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Lumbar dorsal root ganglia (DRG) were harvested from female Wistar rats (140–180 g) and transferred to Dulbecco’s modified Eagle medium (D-MEM, Life Technologies, Gaithersburg, MD) supplemented with 50 µg/mL gentamicin (Sigma). After removal of the connective tissue, ganglia were incubated in collagenase for 75 min (0.28 U/mL; Roche Biochemicals, Mannheim, Germany) and in trypsin for 12 min (25,000 U/mL; Sigma). Cells were dissociated with a fire-polished Pasteur pipette and plated on glass coverslips coated with poly-L-lysine (200 µg/mL; Sigma). Cells were cultured in serum-free TNB 100TM medium (Biochrom, Berlin, Germany), supplemented with penicillin and streptomycin (200 U/mL each; Life Technologies), L-glutamine (2 mM; Life Technologies), and nerve growth factor (mouse NGF 7S, 100 ng/mL; Alomone Labs, Tel Aviv, Israel) in a humid 5% CO2 atmosphere at 37°C for 24–36 h.
Electrophysiological measurements
Current recordings from isolated neurons were performed in external solution (ECS) containing (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose (all from Sigma, Germany), and 10 HEPES (Merck, Darmstadt, Germany) at pH 7.3 adjusted with NaOH. Whole-cell voltage-clamp current measurements were performed at -80 mV holding potential using an Axopatch amplifier and PClamp 6.0 software (Axon Instruments, Union City, CA). Borosilicate electrodes (Science Products, Hofheim, Germany) contained internal solution (ICS, in mM): 148 KCl, 4 MgCl2, 2 Na-ATP (all from Sigma), 0.2 Li-GTP (Calbiochem, Bad Soden, Germany), 0.01 Fura-2 (Molecular Probes, Leiden, Netherlands), and 10 HEPES at pH 7.3 adjusted with KOH. After filling, electrode resistance was 2–4 M
.
For drug application and heat stimulation, a fast 7-channel system with common outlet was used, as given in detail elsewhere (19)
. The application system, placed in close vicinity to the recorded cell (
100 µm), was used for single cell stimulation with controlled heat stimuli or test solutions: IL-1ß or capsaicin. The opening and closing of the solenoid valves for different solutions as well as the DC current for heating were controlled from a switchboard. Recordings were made from capsaicin-sensitive small or medium-sized neurons generally considered to give rise to nociceptive C fibers. The capsaicin sensitivity was tested at the end of the experiment with 1 µM capsaicin for 3 s. All experiments were performed at room temperature and only one neuron was tested per dish.
In the whole-cell configuration of the voltage-clamp technique, Iheat was elicited with ramp-shaped heat stimuli at 60 s intervals (linear rise in temperature from 25°C to 50°C in 5 s). IL-1ß (20 ng/mL), alone or together with pharmacological inhibitors, was used as conditioning intermittent stimulus (90–210 s). For the inhibitors or their negative controls, a 2–3 min pretreatment interval was allowed before starting IL-1ß application.
Calcium measurement in isolated DRG neurons
For calcium measurements, neurons were loaded with the fluorescent dye Fura-2 via the patch pipette after obtaining the whole-cell configuration. Background-corrected fluorescent images were taken with a slow scan CCD camera system with fast monochromator (PTI, Lawrenceville, NJ) coupled to an Axiovert microscope with a 40x fluotar oil immersion objective (Zeiss, Jena, Germany). Fura-2 was excited at 340 and 380 nm wavelengths (
) and fluorescence was collected at = 420 nm at a frequency of 1 Hz, with equal exposure time at each wavelength (200 ms). [Ca2+]i was calculated as previously reported (20)
and calibration constants obtained in vitro were Rmin = 0.44, Rmax = 8.0, and Keff = 1.2 mM.
RT-PCR
Total RNA was isolated from adult female rat DRG’s using RNAzol reagent (WAK-Chemie, Bda-Borchem, Germany) and reverse transcribed into cDNA using MuLV Reverse Transcriptase (Perkin-Elmer, Weiterstadt, Germany). PCR was performed in a 50 µL reaction volume containing 1 x PCR buffer, 1.5 mM MgCl2, 150 µM dNTP, 0.3 µM each specific primer (all from Hybaid Interactiva Biotechnologie, Ulm, Germany) (Table 1
), and 1.25 U AmpliTaq Gold (Perkin-Elmer, Weiterstadt, Germany) under the following conditions: initial denaturation at 94°C, 5 min (1 cycle); 94°C, 45 s, 58°C, 30 s, and 72°C, 45 s (35 cycles), followed by extension at 72°C, 7 min. The amplified fragments were cloned in TOPO vector (Invitrogen, San Diego, CA) and sequenced on the Applied Biosystems 373 DNA sequencer using Taq DyeDeoxy Terminator cycle sequencing kits (Applied Biosystems, Foster City, CA) to confirm the identity of the amplified products.
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In situ hybridization
The IL-1R1 PCR product was used to prepare digoxigenin (DIG)-labeled antisense and sense RNA probes, using T7 RNA polymerase and DIG labeling mix (Boehringer Mannheim, Germany). Lumbar rat DRGs were shock frozen in isopentane cooled in liquid N2. Cryosections (10 µM thick) were cut, fixed (4% phosphate-buffered paraformaldehyde), permeabilized (0.01 M sodium citrate, 0.2 M HCl for 20 min, phosphate-buffered 0.3% Triton X-100 for 5 min, 2 µg/mL proteinase K from Sigma, for 20 min, 37°C), and acetylated (0.1 M triethanolamine containing 0.252% (v/v) acetic anhydride). After prehybridization (2.5% 50xDenhardt’s, 0.05 M EDTA, 0.5 mg/mL yeast tRNA in 50 mM Tris-HCl for 2h at 45°C), the tissue was incubated with 10 µg/mL probe in 0.1 M tris-HCl, 50% deionized formamide, 0.05 M EDTA, 0.25 mg/mL yeast tRNA, 0.5 mg/mL herring sperm DNA, 25% dithiothreitol, 0.002% NaCl, and 10% dextran sulfate (12–16 h, 45°C). Sections were washed in standard sodium citrate buffer, 20 µg/mL RNase A (Sigma; 30 min, 37°C), decreasing concentrations of standard citrate buffer, distilled water (5 min), 0.1 M Tris-HCl (10 min), and 0.1 M maleate buffer (10 min). Detection of the DIG-labeled probe was performed, as recommended by the manufacturer, with alkaline phosphate-conjugated DIG antibody (4°C, 12 h). Color development was allowed to proceed in the dark for 4–16 h. The reaction was terminated by immersion in PBS (pH 7.5). Sections were mounted with Keyser’s glycerylgelatine (Merck).
Reagents
Interleukin 1ß was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Suramin, staurosporine, and bisindolylmaleimide I (BIM1) were from Sigma (St. Louis, MO). Genistein, daidzein, and bisindolylmaleimide V (BIM5) were obtained from Calbiochem (San Diego, CA). Stock solutions were prepared in DMSO (final DMSO concentration 0.1% or less) or distilled water. All solutions were diluted in ECS to the final concentration immediately before the experiment.
Statistical evaluation
For data analysis, the Statistica Software package Statsoft 5.0 was used. All summarizing values are given as means ± SE. Mean Iheat amplitudes before and after cytokine stimulation were calculated and intraindividual comparisons were performed using the Wilcoxon matched pairs test. To analyze the heat threshold shift toward lower temperatures after IL-1ß treatment, current values were plotted against temperature in every neuron. For a global analysis, we averaged the Iheat values corresponding to every °C temperature, and the Wilcoxon matched pairs test was used for statistical analysis. To compare the effects of different inhibitors on IL-1ß-induced sensitization of Iheat, the actual current values were divided by the Iheat value just before stimulation. For interindividual comparisons, the Mann-Whitney U test was performed using the normalized data described above. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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a). However, after cytokine stimulation, an increase in the amplitude of Iheat was detected (Fig. 1a
): in 7 of 11 neurons (63.64%), 20 ng/mL IL-1ß induced a significant increase in the amplitude of Iheat from 466 ± 198 pA to 914 ± 218 pA, P < 0.05 (Fig. 1b
). In 4 of 7 neurons, 2 ng/mL was also effective but the effect was less pronounced (data not shown). Therefore, 20 ng/mL was used for all further pharmacological experiments. To compare the heat thresholds, current values before and after IL-1ß application were plotted against temperature in each individual cell (Fig. 1c
). Exposure to IL-1ß 20 ng/mL induced a significant drop of heat threshold of 3°C after cytokine stimulation (Fig. 1d
). These IL-1 effects were similar in the presence of suramin used to inhibit G-protein-coupled receptors. Overall, IL-1ß induced a 2.72-fold increase in Iheat, which was not significantly modified in the presence of suramin 100 µM (2.6-fold increase in Iheat; n=6; Fig. 5a
).
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Involvement of tyrosine kinases
The brief exposure time (90–210 s) needed to elicit the effect suggests a fast signaling cascade. Protein kinases are recruited by IL-1ß binding to IL-1RI; to investigate whether protein kinases were involved, the nonspecific protein kinase inhibitor staurosporine (100 nM) was used. In the presence of staurosporine, neurons showed only a minor increase in Iheat amplitude after IL-1ß treatment (443±46 pA before vs. 530±41 pA after IL-1ß; n=6; Fig. 2
a, b). Staurosporine also prevented the IL-1ß-induced drop in heat threshold (Fig. 2c, d
). Among protein kinases, the PTK are likely to be involved in IL-1ß signaling. Hence, the specific PTK inhibitor genistein (10 µM) was used; in the presence of genistein, neurons no longer exhibited increased Iheat amplitude after application of IL-1ß (402±50 pA before vs. 494±78 pA after IL-1ß; n=11; Fig. 3
a, b). The heat threshold in the presence of genistein was unchanged (Fig. 3c, d
). In the presence of the negative control daidzein, IL-1ß effects were similar to controls. As depicted in Fig. 5c
, the relative increase in Iheat induced by IL-1ß (2.72-fold) was significantly reduced by genistein (1.28-fold; P<0.05; n=11) but not by daidzein (2.16-fold; n=6; Fig. 5
b).
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Involvement of PKC
Another family of protein kinases involved in downstream IL-1RI signaling are isoforms of PKC, which are also blocked by staurosporine. To differentiate between PTK and PKC effects the specific PKC inhibitor BIM1 200 nM was used. This prevented the IL-1ß-induced facilitation of Iheat (424±65 pA before vs. 504±59 pA after IL-1ß; n=8; Fig. 4
a, b) and the drop in heat threshold after IL-1ß treatment (Fig. 4c, d
). In the presence of the negative control BIM5, sensitization was similar to that obtained under control conditions (2.3-fold; n=7) whereas it was significantly reduced by BIM1 (1.17-fold; P<0.01; n=8, Fig. 5
c).
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IL-1RI but not RII expression in rat DRG neurons
To determine what receptor subtype underlies the sensitizing effect of IL-1ß, total mRNA was isolated from rat DRG and RT-PCR revealed IL-1RI but not RII expression (Fig. 6
a). To address whether IL-1RI-mRNA was located in neurons or in non-neuronal cells, we performed a nonradioactive in situ hybridization in DRG sections using a sequence-specific RNA probe for IL-1RI. We could detect mRNA for IL-1RI in the majority of the neurons, including small and medium-sized ones (Fig. 6b-d
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using an electrophysiological approach, we document that brief application of IL-1ß significantly and rapidly potentiates Iheat in sensory neurons. This explains the cellular mechanisms underlying previous in vivo and in vitro reports of nociceptor sensitization to noxious heat (14
15)
. No excitatory inward currents were obtained in response to the cytokine itself, and this corroborates the lack of nociceptor excitation in a previous in vitro study (15)
. Spontaneous excitatory activity observed in vivo after local application of IL-1ß is therefore likely due to indirect effects involving the complexity of the living tissue (14)
.
IL-1ß binds to specific cell surface receptors IL-1RI and IL-1RII, belonging to the large Toll-like receptor superfamily (21)
. The IL-1RI but not IL-1RII according to our and previous data is expressed in sensory neurons (17)
. Whereas IL-1RI transduces the biological effects of IL-1ß, IL-1RII serves as a decoy receptor restricting the effect of the cytokine on its target cells (22)
. When IL-1ß binds to IL-1RI, an accessory protein (IL-1RAcP) is attracted and an adapter protein MyD88 is recruited to the IL-1RI/IL-1RAcP complex, which activates an IL-1R-associated kinase (IRAK) (23
24
25
26)
. IRAK interacts with TNF receptor-associated factor 6 (TRAF6) to activate the nuclear signal NF-
B and several kinases (27
28
29
30)
. How IL-1ß after IL-1RI activation triggers its fast effects in sensory neurons is so far unknown. It was reported that IL-1ß early activates tyrosine kinases, probably downstream of IRAK (31
32
33)
. The present study demonstrates that neurons in the presence of the PTK inhibitor genistein no longer exhibit potentiation of Iheat after IL-1ß treatment. PTK may phosphorylate the heat-transducing mechanism in nociceptors. Indeed, the amino acid structure of the heat transducer VR-1 contains at least four consensus sites for PTK phosphorylation at Y351, Y565, Y631, and Y666. Alternatively, PTK may activate downstream signaling in the target cell, e.g., certain PKC isoenzymes. In our experiments, the acute IL-1ß-induced sensitization was prevented by the PTK inhibitor but also by staurosporine, a rather nonselective protein kinase inhibitor with preferential inhibition of PKCs, and by the PKC selective BIM1. This indeed suggests IL-1RI signaling through PTK-mediated downstream activation of PKC.
PKC-dependent potentiation of Iheat is also known for the inflammatory mediator bradykinin (BK) (34
35
36
37
38)
. BK-induced effects depended on PKC
, although some other PKC isoenzymes are expressed in rat sensory neurons (39)
and PKC-/- mice showed an attenuated thermal hyperalgesia (40)
. A possible target of PKC-mediated phosphorylation in the IL-1ß signaling pathway could be heat-transducing vanilloid receptors like VR-1 or VRL-1. VR-1-expressing HEK293 cells exhibited a PKC-induced potentiation of the inward currents elicited by heat, capsaicin, protons, and anadamide (41)
. In rat nociceptors and VR-1-expressing oocytes, VR-1 channel activity could be activated and facilitated by PKC (42)
. Since VR-1 not only is integrated in the soma membrane or the membrane of peripheral nerve terminals but is also functionally expressed along the nociceptor axon and the nervi nervorum (43
, 44)
, the IL-1ß-mediated activation of PKC with VR-1 interaction may well explain the role of cytokines in spinal neuroinflammation and neuropathy (4
, 7)
.
Since the application of IL-1ß did not result in rises in intracellular calcium levels, one needs to assume that calcium-independent PKC isoenzymes are involved in the potentiation process reported here similar to those observed previously (45)
. Of the calcium-independent PKC isoforms, PKC, PKC
, and PKC
are expressed in rat DRG and may contribute to IL-1ß-induced heat sensitization (39)
.
In summary, our results represent the first evidence that brief exposure to IL-1ß directly sensitizes rat sensory neurons to heat, probably via the type 1 of IL-1 receptors. The suggested mechanism uses recruitment of PTK and downstream activation of PKC, which phosphorylates heat transducer molecules like VR-1 to cause heat hyperalgesia (Fig. 7
).
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ACKNOWLEDGMENTS |
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Received for publication February 18, 2002.
Revision received June 5, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-1ß and IL-6 but not IL-8 in the development of heat hyperalgesia: effects on heat-evoked calcitonin gene-related peptide release from rat skin. J. Neurosci. 20,6289-6293This article has been cited by other articles:
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  C. E. Constantin, N. Mair, C. A. Sailer, M. Andratsch, Z.-Z. Xu, M. J. F. Blumer, N. Scherbakov, J. B. Davis, H. Bluethmann, R.-R. Ji, et al. Endogenous Tumor Necrosis Factor {alpha} (TNF{alpha}) Requires TNF Receptor Type 2 to Generate Heat Hyperalgesia in a Mouse Cancer Model J. Neurosci., May 7, 2008; 28(19): 5072 - 5081. [Abstract] [Full Text] [PDF]
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 K. K. Rau, N. Jiang, R. D. Johnson, and B. Y. Cooper Heat Sensitization in Skin and Muscle Nociceptors Expressing Distinct Combinations of TRPV1 and TRPV2 Protein J Neurophysiol, April 1, 2007; 97(4): 2651 - 2662. [Abstract] [Full Text] [PDF]
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