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. 2010 Feb 10;30(6):2138-49.
doi: 10.1523/JNEUROSCI.5752-09.2010.

GRK2: a novel cell-specific regulator of severity and duration of inflammatory pain

Niels Eijkelkamp  1 , Cobi J Heijnen, Hanneke L D M Willemen, Ronald Deumens, Elbert A J Joosten, Wendy Kleibeuker, Ilona J M den Hartog, Cindy T J van Velthoven, Cora Nijboer, Mohammed A Nassar, Gerald W Dorn 2nd, John N Wood, Annemieke Kavelaars
Affiliations

GRK2: a novel cell-specific regulator of severity and duration of inflammatory pain

Niels Eijkelkamp et al. J Neurosci. .

Abstract

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms have only begun to be unraveled. GRK2 regulates cellular signaling by promoting G-protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2(+/-) mice with an approximately 50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using Cre-Lox technology. Carrageenan- or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Na(v)1.8 nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitization. In vivo, CCL3-induced acute hyperalgesia in GRK2(+/-) mice was mediated via TRPV1. Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2(+/-) mice was associated with ongoing microglial activation and increased phospho-p38 and tumor necrosis factor alpha (TNF-alpha) in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-alpha activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2(+/-) mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the pathophysiological relevance of microglial GRK2. Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia.

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Figures

Figure 1.
Figure 1.
Reduced GRK2 increases and prolongs inflammation-induced thermal hyperalgesia and mechanical allodynia. a, GRK2 protein level in dorsal root ganglia (n = 4) and spinal cord (n = 8) of GRK2+/− and WT animals as determined by Western blot analysis. The inset shows representative Western blots for GRK2 with α-tubulin as a loading control. b, Percentage decrease in heat withdrawal latency after intraplantar carrageenan injection in WT (n = 8) and GRK2+/− mice (n = 8). c, Mechanical allodynia induced by intraplantar carrageenan injection in WT (n = 4) and GRK2+/− (n = 4) mice. Mechanical sensitivity was determined using a calibrated von Frey hair that does not elicit a withdrawal response in naive control mice. Data represent the percentage of withdrawal responses to six applications per paw. d, Percentage decrease in heat withdrawal latency after intraplantar CCL3 injection in WT (n = 11) and GRK2+/− mice (n = 11). e, Mechanical allodynia induced by intraplantar injection in WT (n = 4) and GRK2+/− (n = 4) mice. f, Percentage decrease in heat withdrawal latency induced by intraplantar IL-1β in WT (n = 14) and GRK2+/− mice (n = 14). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 2.
Figure 2.
GRK2 does not affect carrageenan- or CCL3-induced inflammatory response. a, Carrageenan-induced increase in paw thickness expressed as percentage of baseline (n = 8). b, MPO content of paw biopsies as a measure of granulocyte infiltration at 6 h, 1 d, and 7 d after carrageenan injection (n = 6 − 12). c, IL-1β mRNA in paw biopsies at 6 h after carrageenan injection. d, CCL3 mRNA in paw biopsies at 6 h after carrageenan injection (n = 7). e, IL-1β mRNA in paw biopsies at 24 h after intraplantar CCL3 injection (n = 4). f, TNF-α mRNA in paw biopsies at 24 h after CCL3 injection (n = 4). g, CCL3-induced increase in paw thickness expressed as a percentage of baseline (n = 7). h, MPO content of paw biopsies at 6 h after intraplantar CCL3 injection (n = 8 − 11). Data are expressed as mean ± SEM. **p < 0.01.
Figure 3.
Figure 3.
Reduced GRK2 in Nav1.8+ sensory neurons increases acute hyperalgesia. a, GRK2 protein levels in dorsal root ganglia (n = 4) and spinal cord (n = 4) of Nav1.8-GRK2f/+ and control Nav1.8-GRK2+/+ mice. The inset shows Western blots for GRK2 with α-tubulin as a loading control. b, Percentage decrease in heat withdrawal latency after intraplantar carrageenan injection in Nav1.8-GRK2f/+ (n = 8) mice and in Nav1.8-GRK2+/+ controls (n = 8). c, Percentage decrease in heat paw withdrawal latency after intraplantar CCL3 injection in Nav1.8-GRK2f/+ mice (n = 16) and in Nav1.8-GRK2+/+ controls (n = 16). d, Percentage decrease in heat withdrawal latency after intraplantar IL-1β injection in Nav1.8-GRK2f/+ mice (n = 8) and Nav1.8-GRK2+/+ controls (n = 8). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4.
Figure 4.
Regulation of CCL3-induced TRPV1 sensitization by GRK. a, CCL3-induced (100 ng/ml) Ca2+ response in F11 cells expressing CCR1 treated with GRK2 siRNA to reduce GRK2 expression or control nonspecific siRNA (scr). The inset shows a reduction in GRK2 protein. b, Capsaicin-induced Ca2+ response in F11 cells overexpressing TRPV1 and CCR1 treated with GRK2 siRNA or control nonspecific siRNA (scr). Cells were pretreated with or without CCL3 (100 ng/ml) for 20 min to induce TRPV1 sensitization. Capsaicin was added and remained in the medium during the entire period of measurement. c, Quantitative analysis of CCL3-induced sensitization of TRPV1 to capsaicin in cells treated with GRK2 siRNA or control nonspecific siRNA (scr). Each bar represents the mean Fura-2 ratio of 15 responding cells as determined during a 100 s recording period in two independent experiments. d, CCL3-induced (100 ng/ml) Ca2+ response in F11 cells expressing CCR1 with or without [empty vector (EV)] GRK2 overexpression. The inset shows GRK2 expression. e, Capsaicin-induced Ca2+ response in F11 cells expressing TRPV1 and CCR1 and overexpressing GRK2 or transfected with control vector (EV). Cells were pretreated with or without CCL3 for 20 min. f, Quantitative analysis capsaicin-induced Ca2+ response in F11 cells expressing TRPV1 and CCR1 overexpressing GRK2 or transfected with empty vector (EV). Cells were pretreated with or without CCL3 for 20 min. Bars represent the mean Fura-2 ratio as determined during a 100 s recording period of 40 responding cells measured in three independent experiments. g, The TRPV1 antagonist capsazepine was injected intraplantarly 30 min before measurement of heat withdrawal latency 2 h after CCL3 injection. Thermal hyperalgesia is depicted as the percentage of decrease in heat withdrawal latency (n = 8 − 12). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5.
Figure 5.
GRK2 in microglia/macrophages regulates chronic hyperalgesia. a, GRK2 protein levels in microglia (n = 4), macrophages (n = 3), and astrocytes (n = 4) of LysM-GRK2f/+ and control LysM-GRK2+/+ mice. The inset depicts Western blots for GRK2 protein and β-actin as a loading control. b, Percentage decrease in heat withdrawal latency after intraplantar carrageenan injection in LysM-GRK2f/+ (n = 8) and control LysM-GRK2+/+ (n = 8) mice. c, Percentage decrease in heat withdrawal latency after intraplantar CCL3 injection in control LysM-GRK2+/+ (n = 8) and in LysM-GRK2f/+ mice (n = 8). d, Percentage decrease in heat withdrawal latency after intraplantar IL-1β injection in control LysM-GRK2+/+ (n = 8) and LysM-GRK2f/+ mice (n = 8). e, No effect of PMN depletion on CCL3-induced thermal hyperalgesia in LysM-GRK2f/+ (n = 8) and control LysM-GRK2+/+ (n = 8) mice. f, No effect of PMN depletion on carrageenan-induced thermal hyperalgesia in LysM-GRK2f/+ (n = 8) and control LysM-GRK2+/+ (n = 8) mice. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 6.
Figure 6.
Effect of reduction of GRK2 in GFAP-positive astrocytes on carrageenan- and CCL3-induced hyperalgesia. a, GRK2 protein levels in microglia (n = 4) and astrocytes (n = 4) of GFAP-GRK2f/+ and control GFAP-GRK2+/+ mice. The inset depicts Western blots for GRK2 protein and β-actin as a loading control. b, Percentage decrease in heat withdrawal latency after intraplantar carrageenan injection in GFAP-GRK2+/+ (n = 8) and GFAP-GRK2f/+ mice (n = 8). c, Percentage decrease in heat withdrawal latency after intraplantar CCL3 injection in GFAP-GRK2+/+ (n = 16) and GFAP-GRK2f/+ (n = 16). Data are expressed as mean ± SEM.
Figure 7.
Figure 7.
Microglial/macrophage activity is required for chronic carrageenan-induced hyperalgesia. a, WT and GRK2+/− mice received an intraplantar injection of carrageenan or saline. At 2 d after injection, spinal cord was collected, and frozen sections were stained with Iba-1 to visualize microglia. Representative example of lumbar segment L2 of one of three mice per group is displayed. b, WT and GRK2+/− mice were treated with minocycline or PBS starting 1 d before intraplantar injection of carrageenan (n = 8 per group), and heat withdrawal latencies were determined. c, WT and GRK2+/− mice were treated with two intrathecal injections (6 h interval) of minocycline or PBS at day 7 after carrageenan injection. Heat withdrawal latencies were determined 1 h before and 24 h after administration of minocycline (n = 6). Data are expressed as mean ± SEM. **p < 0.01; ***p < 0.001.
Figure 8.
Figure 8.
Role of spinal p38 activation in carrageenan-induced chronic hyperalgesia. a, LPS-induced TNF-α production by LysM-GRK2+/+ WT and LysM-GRK2f/+ microglia. Primary cultures of WT and GRK2+/− microglia were stimulated for 3 h with LPS in presence or absence of the p38 inhibitor SB203580 (20 μm). TNF-α in the supernatant was determined by ELISA. TNF-α levels were undetectable in unstimulated microglia. Data are from two independent experiments performed in triplicate. b, Western blot analysis of p-p38 levels in lumbar spinal cord of saline- or carrageenan-treated WT and GRK2+/− mice at 7 d after intraplantar injection. c, WT and GRK2+/− mice were treated with an intrathecal injection of the p38 inhibitor SB239063 (5 μg/mouse) at day 7 after intraplantar carrageenan injection. Heat withdrawal latencies were determined 1 h before and 2 h after administration of SB239063 (n = 4). d, WT and GRK2+/− were treated with an intrathecal injection of etanercept (100 μg/mouse) at day 7 after intraplantar carrageenan injection and heat withdrawal latencies were determined (n = 6). e, TNF-α levels in spinal segments L1–L5 (lumbar) or T1–T8 (thoracic) of GRK2+/− mice were determined 7 d after intraplantar vehicle or carrageenan injection. The effect of p38 inhibition on carrageenan-induced TNF-α was tested 7 d after intraplantar carrageenan by treating mice intrathecally with SB239063 24 and 2 h before collection of spinal cords. Data represent the level of TNF-α expressed as percentage of vehicle-treated controls. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 9.
Figure 9.
Spinal cord microglial/macrophage GRK2 levels after SNT. a, The sensitivity to mechanical stimulation was determined in sham-operated rats or rats subjected to a unilateral L5 SNT two weeks after surgery (n = 8 per group). b, Scatter plot and bar graph of GRK2 levels in OX-42+ microglia/macrophages isolated from ipsilateral and contralateral lumbar spinal cord of sham-operated and SNT rats at day 14 after surgery. GRK2 expression was quantified in 50–100 cells on three separate slides containing cells isolated from two to three rats per slide. c, Representative pictures of GRK2, OX-42, and DAPI staining of isolated spinal cord microglia/macrophages from ipsilateral and contralateral lumbar spinal cord of sham-operated and SNT rats (first 4 columns). Specificity of GRK2 staining was determined by administration of a specific blocking peptide to the primary antibody and for OX-42 by using an isotype control antibody and is shown in the two columns at the right. Data are expressed as mean ± SEM. ***p < 0.001.
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