Microglial/macrophage GRK2 determines duration of peripheral IL-1beta-induced hyperalgesia: contribution of spinal cord CX3CR1, p38 and IL-1 signaling - PubMed (original) (raw)

Microglial/macrophage GRK2 determines duration of peripheral IL-1beta-induced hyperalgesia: contribution of spinal cord CX3CR1, p38 and IL-1 signaling

Hanneke L D M Willemen et al. Pain. 2010 Sep.

Abstract

Chronic pain associated with inflammation is a major clinical problem, but the underlying mechanisms are incompletely understood. Recently, we reported that GRK2(+/-) mice with a approximately 50% reduction of GRK2 develop prolonged hyperalgesia following a single intraplantar injection of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). Here we show that spinal microglia/macrophage GRK2 is reduced during chronic inflammation-induced hyperalgesia. Next, we applied CRE-Lox technology to create mice with low GRK2 in microglia/macrophages/granulocytes (LysM-GRK2(f/+)), or sensory neurons or astrocytes. Only mice deficient in microglial/macrophage/granulocyte GRK2 display prolonged IL-1beta-induced hyperalgesia that lasts up to 8days. Two days after intraplantar IL-1beta, increased microglial/macrophage activity occurs in the lumbar but not thoracic spinal cord of GRK2-deficient mice. Intrathecal pre-treatment with minocycline, an inhibitor of microglia/macrophage activation, accelerates resolution of hyperalgesia independent of genotype and prevents transition to chronic hyperalgesia in GRK2(+/-) mice. Ongoing hyperalgesia in GRK2(+/-) mice is reversed by minocycline administration at days 1 and 2 after IL-1beta injection. Similarly, IL-1beta-induced hyperalgesia in LysM-GRK2(f/+) mice is attenuated by intrathecal administration of anti-CX3CR1 to abrogate fractalkine signaling, the p38 inhibitor SB239063 and the IL-1 antagonist IL-1ra. These data establish that chronic inflammatory hyperalgesia is associated with reduced GRK2 in microglia/macrophages and that low GRK2 in these cells is sufficient to markedly prolong hyperalgesia after a single intraplantar injection of IL-1beta. Ongoing hyperalgesia is maintained by spinal microglial/macrophage activity, fractalkine signaling, p38 activation and IL-1 signaling. We propose that chronic inflammation decreases spinal microglial/macrophage GRK2, which prevents silencing of microglia/macrophage activity and thereby contributes to prolonged hyperalgesia.

Copyright (c) 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

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Conflict of interest statement

We have no conflicts of interests related to this study.

Figures

Figure 1

Figure 1. GRK2 levels in spinal microglia/macrophages during inflammatory hyperalgesia

(a) GRK2 expression in microglia/macrophages isolated from lumbar spinal cord at 6 days after intraplantar carrageenan or saline administration was compared by immunofluorescence analysis. Representative pictures of GRK2, CD-11b and DAPI-staining of isolated microglia/macrophages. (b) GRK2 expression was quantified in approximately 60 cells per group on three different slides each containing microglia/macrophages from spinal cord of 4 mice per group. Data are expressed as mean SEM. *** p < 0.001.

Figure 2

Figure 2. GRK2 regulates duration of peripheral IL-1β-induced hyperalgesia

Percentage decrease in heat withdrawal latency in WT and GRK2+/− mice (a) after intraplantar IL-1β at a dose of 1 ng (n = 8) (b) 4 hours after intraplantar injection of 0 (saline), 0.1 or 1 ng IL-1β (n = 8). Data are expressed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Figure 3

Figure 3. Gene dosage effect and cellular specificity of the effect of low GRK2 on thermal hyperalgesia and mechanical allodynia

(a) GRK2 expression in primary microglia and in CD11b+ peritoneal macrophages from LysM-GRK2+/+ (WT), LysM-GRK2f/+ and LysM-GRK2f/f mice was determined by Western blot analysis. (b) Percentage decrease in heat withdrawal latency after intraplantar IL-1β (1 ng) injection in control LysM-GRK2+/+ (WT) and LysM-GRK2f/+ mice (n = 8) (c) Change in heat withdrawal latency in control LysM-GRK2+/+ (WT) (n = 18), heterozygous LysM-GRK2f/+ (n = 12) and homozygous LysM-GRK2f/f mice (n = 14) after intraplantar injection of 1 ng IL-1β. (d) Decrease in 50% threshold for withdrawal in response to mechanical stimulation after intraplantar IL-1β (1 ng) in LysM-GRK2+/+ (WT) and LysM-GRK2f/+ mice (n = 8). (e) MPO-content of paw biopsies as a measure of neutrophil infiltration at 2 days after intraplantar IL-1β injection (n = 8). (f–g) As a measure of macrophage infiltration after intraplantar IL-1β adminstration, samples were analyzed for (f) CX3CR1 mRNA expression in paw biopsies at 0, 6 and 48 hours after injection of IL-1β (n = 6) (g) Iba-1 staining in paw biopsies at 2 days after injection of IL-1β. Data are expressed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4

Figure 4. Contribution of GRK2 in astrocytes and primary sensory neurons to IL-1β-induced hyperalgesia

Percentage decrease in heat withdrawal latency after intraplantar IL-1β (1 ng) in (a) control GFAP-GRK2+/+ (WT) and GFAP-GRK2f/+ mice (n = 8) (b) control Nav1.8-GRK2+/+ (WT) and Nav1.8-GRK2f/+ mice (n = 8).

Figure 5

Figure 5. Spinal cord microglia/macrophage activity

(a) LysM-GRK2+/+ (WT) and LysM-GRK2f/+ mice received an intraplantar injection of IL-1β or saline. At two days after injection, spinal cord was collected and frozen sections of lumbar spinal cord (L2–L4) and as a control thoracic spinal cord (T8–T10) were stained with Iba-1 to visualize microglia/macrophages. Representative example of morphology of Iba-1-positive cells in the dorsal horn (see drawing for exact location) of one out of three mice per group is displayed. (b–d) WT (white bars) and GRK2+/− mice (grey bars) received an intraplantar injection of IL-1β (1ng) and lumbar and as a control thoracic spinal cord was collected two days later. Samples were analysed for mRNA encoding (b) IL-1β; (c) Cathepsine S; (d) CX3CR1; and (e) fractalkine. n = 10 per group. * p<0.05

Figure 6

Figure 6. Effect of minocycline treatment at day one and two after intraplantar IL-1β on hyperalgesia

WT and GRK2+/− mice received an intraplantar injection of IL-1β (1 ng) and the percentage decrease in heat withdrawal latency was determined. At one and two days after IL-1β, minocycline (50 mg/kg) or vehicle was administered (n = 4 per group). Data are expressed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 vs. vehicle.

Figure 7

Figure 7. Factors contributing to ongoing hyperalgesia in GRK2-deficient mice

LysM-GRK2f/+ mice received an intraplantar injection of IL-1β and the percentage decrease in heat withdrawal latency was determined. At day 2 after intraplantar IL-1β, mice received an intrathecal injection of (a) αCX3CR1 (1 ug/mouse) or rabbit IgG (1 μg/mouse) (n = 6 per group) (b) p38 inhibitor SB239063 (5 μg/mouse; n = 8) or vehicle (n = 4) (c) PI3 kinase inhibitor LY294002 (5 μg/mouse) or vehicle (n = 4 per group) (d) IL-1ra (10 ng/mouse; n = 6) or vehicle (n = 4). Heat sensitivity in IL-1β-treated LysM-GRK2+/+ (WT) mice had already returned to baseline at day 2 after injection and was not affected by any of the treatments (data not shown). The inhibitors did not affect heat sensitivity in naïve mice of either genotype. Data are expressed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 vs. vehicle or control IgG.

Figure 8

Figure 8. Contribution of early spinal cord microglial/macrophage activity to IL-1β-induced hyperalgesia

WT and GRK2+/− mice (n = 8 per group) received an intrathecal injection of minocycline (30 μg) or vehicle 60 min. prior to intraplantar IL-1β and the percentage change in heat withdrawal latency was determined. Data are expressed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 for GRK2+/− mice/minocycline vs. GRK2+/− mice/vehicle. ## p<0.01 for both genotypes with minocycline vs. saline.

Figure 9

Figure 9. Contribution of fractalkine to prolonged IL-1β-induced hyperalgesia in GRK2-deficient mice

(a) Mice received an intrathecal injection of fractalkine (1 ng) and the percentage decrease in heat withdrawal latency was determined in LysM-GRK2+/+ (WT) and in LysM-GRK2f/+ mice (n = 8 per group). (b–c) Mice received and intraplantar injection of IL-1β. The effect of intrathecal administration of anti-CX3CR1 (1 μg/mouse) or rabbit IgG (1 μg/mouse) (b) before or (c) 4 hours after intraplantar IL-1β administration on hyperalgesia in LysM-GRK2+/+ (WT) and LysM-GRK2f/+ mice (n = 4 per group) was determined. Data are expressed as mean ± SEM. *** p<0.001.

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