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Genetic evidence for involvement of neuronally expressed S1P₁ receptor in nociceptor sensitization and inflammatory pain.

Mair N, Benetti C, Andratsch M, Leitner MG, Constantin CE, Camprubí-Robles M, Quarta S, Biasio W, Kuner R, Gibbins IL, Kress M, Haberberger RV - PLoS ONE (2011)

Bottom Line: Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue.We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors.Our data show that neuronally expressed S1P₁ receptors play a significant role in regulating nociceptor function and that S1P/S1P₁ signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

View Article: PubMed Central - PubMed

Affiliation: Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria. norbert.mair@i-med.ac.at

ABSTRACT
Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P₁ receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P₁ receptor. Our data show that neuronally expressed S1P₁ receptors play a significant role in regulating nociceptor function and that S1P/S1P₁ signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

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Expression of S1P1 receptors in sensory neurons.(A) S1P receptor mRNA expression was detected with reverse transcription PCR in DRG explants. (B) Quantitative real-time PCR revealed expression of S1P1, S1P2 and S1P3 mRNA in DRG explants (total), acutely isolated neurons (acute) and 1-day-old cultures (1 d) (n = 5 experiments). In contrast, S1P4 and S1P5 mRNA levels were lower in DRG explants and absent in isolated neurons. (C) Immunoreactivity for S1P1 was present in neurons and intraganglionic capillaries (arrowhead). S1P1-IR was colocalized with immunoreactivity for peripherin, whereas S1P1-IR was absent in NF200-positive neurons. Scale bars  = 50 µm. (D) S1P1 receptor colocalized with the small neuron marker I-B4 in the vast majority of cultured neurons but usually not with CGRP or Nf200, a marker for myelinated neurons (n = 4 experiments, scale bars  = 20 µm). (E) Size distribution of S1P1-IR positive neurons revealed that S1P1-IR expressing cells are amongst the small diameter neurons (n = 6 experiments, 304 neurons). Only 2% of S1P1-IR+ neurons had diameters >20 µm. (F) Expression of S1P1 immunoreactivity was absent after preabsorption of the antibodies with the corresponding peptide.
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pone-0017268-g003: Expression of S1P1 receptors in sensory neurons.(A) S1P receptor mRNA expression was detected with reverse transcription PCR in DRG explants. (B) Quantitative real-time PCR revealed expression of S1P1, S1P2 and S1P3 mRNA in DRG explants (total), acutely isolated neurons (acute) and 1-day-old cultures (1 d) (n = 5 experiments). In contrast, S1P4 and S1P5 mRNA levels were lower in DRG explants and absent in isolated neurons. (C) Immunoreactivity for S1P1 was present in neurons and intraganglionic capillaries (arrowhead). S1P1-IR was colocalized with immunoreactivity for peripherin, whereas S1P1-IR was absent in NF200-positive neurons. Scale bars  = 50 µm. (D) S1P1 receptor colocalized with the small neuron marker I-B4 in the vast majority of cultured neurons but usually not with CGRP or Nf200, a marker for myelinated neurons (n = 4 experiments, scale bars  = 20 µm). (E) Size distribution of S1P1-IR positive neurons revealed that S1P1-IR expressing cells are amongst the small diameter neurons (n = 6 experiments, 304 neurons). Only 2% of S1P1-IR+ neurons had diameters >20 µm. (F) Expression of S1P1 immunoreactivity was absent after preabsorption of the antibodies with the corresponding peptide.

Mentions: To date, five metabotropic S1P1–5 receptors for S1P have been identified which are members of the edg (endothelial differentiation gene) family of G-protein coupled receptors (GPCRs) [32]–[35]. To elucidate which S1P receptors were relevant for the regulation of thermal sensitivity in sensory neurons, we performed receptor mRNA expression profile analyses in DRG explants, in acutely dissociated neurons, and in primary DRG neuron enriched cultures. Quantitative PCR showed the following rank order of relative receptor mRNA expression for DRG explants: S1P3 > S1P1 > S1P2 > S1P4 > S1P5 (Fig. 3A, B). These explants contain neurons and non-neuronal cells including satellite cells, myelinating and non-myelinating Schwann cells, resident immune cells, endothelial cells and vascular immune cells [36]. S1P receptors have been found expressed in most of these cell types (for review see [37]. However, in preparations enriched with acutely isolated sensory neurons (<1% non-neuronal cells) and in 1-day-old neuron-enriched cultures mRNAs for S1P1, S1P2 and S1P3 but not S1P4 and S1P5 receptors were expressed. The relative expression levels of mRNA for S1P1–3 receptors were similar in acutely dissociated neurons and in 1-day-old cultures (Fig. 3A, B). Together this suggests that S1P1, S1P2 and S1P3 receptor subtypes were expressed in sensory neurons whilst S1P4 and S1P5 subtypes were not. We conclude, therefore, that in the explant preparations, mRNAs for S1P4 and S1P5 receptors were probably derived from non-neuronal cells within the DRG. Most nociceptive neurons are small to medium-sized peripherin-positive neurons [38]. They include glial-derived neurotrophic factor (GDNF)/protein typrosine kinase receptor (c-RET) dependent neurons with affinity of the isolectin B4 (I-B4+) and NGF/tyrosine kinase A (TrkA) dependent neurons that contain the neuropeptides calcitonin-gene related peptide (CGRP) and substance P [39]. Immunoreactivity for S1P1 receptor was found predominantly in small peripherin-IR neurons in DRG sections (Fig. 3C, D, E, F). In sections and neuron cultures, S1P1 immunoreactivity predominantly occurred in small I-B4+, Nf200-negative neurons. A smaller group of CGRP-IR neurons also displayed immunoreactivity for S1P1 (Table 1). Based on this co-expression profile, we predicted that the actions of S1P on nociceptor function were most likely mediated by the S1P1 receptor.


Genetic evidence for involvement of neuronally expressed S1P₁ receptor in nociceptor sensitization and inflammatory pain.

Mair N, Benetti C, Andratsch M, Leitner MG, Constantin CE, Camprubí-Robles M, Quarta S, Biasio W, Kuner R, Gibbins IL, Kress M, Haberberger RV - PLoS ONE (2011)

Expression of S1P1 receptors in sensory neurons.(A) S1P receptor mRNA expression was detected with reverse transcription PCR in DRG explants. (B) Quantitative real-time PCR revealed expression of S1P1, S1P2 and S1P3 mRNA in DRG explants (total), acutely isolated neurons (acute) and 1-day-old cultures (1 d) (n = 5 experiments). In contrast, S1P4 and S1P5 mRNA levels were lower in DRG explants and absent in isolated neurons. (C) Immunoreactivity for S1P1 was present in neurons and intraganglionic capillaries (arrowhead). S1P1-IR was colocalized with immunoreactivity for peripherin, whereas S1P1-IR was absent in NF200-positive neurons. Scale bars  = 50 µm. (D) S1P1 receptor colocalized with the small neuron marker I-B4 in the vast majority of cultured neurons but usually not with CGRP or Nf200, a marker for myelinated neurons (n = 4 experiments, scale bars  = 20 µm). (E) Size distribution of S1P1-IR positive neurons revealed that S1P1-IR expressing cells are amongst the small diameter neurons (n = 6 experiments, 304 neurons). Only 2% of S1P1-IR+ neurons had diameters >20 µm. (F) Expression of S1P1 immunoreactivity was absent after preabsorption of the antibodies with the corresponding peptide.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040773&req=5

pone-0017268-g003: Expression of S1P1 receptors in sensory neurons.(A) S1P receptor mRNA expression was detected with reverse transcription PCR in DRG explants. (B) Quantitative real-time PCR revealed expression of S1P1, S1P2 and S1P3 mRNA in DRG explants (total), acutely isolated neurons (acute) and 1-day-old cultures (1 d) (n = 5 experiments). In contrast, S1P4 and S1P5 mRNA levels were lower in DRG explants and absent in isolated neurons. (C) Immunoreactivity for S1P1 was present in neurons and intraganglionic capillaries (arrowhead). S1P1-IR was colocalized with immunoreactivity for peripherin, whereas S1P1-IR was absent in NF200-positive neurons. Scale bars  = 50 µm. (D) S1P1 receptor colocalized with the small neuron marker I-B4 in the vast majority of cultured neurons but usually not with CGRP or Nf200, a marker for myelinated neurons (n = 4 experiments, scale bars  = 20 µm). (E) Size distribution of S1P1-IR positive neurons revealed that S1P1-IR expressing cells are amongst the small diameter neurons (n = 6 experiments, 304 neurons). Only 2% of S1P1-IR+ neurons had diameters >20 µm. (F) Expression of S1P1 immunoreactivity was absent after preabsorption of the antibodies with the corresponding peptide.
Mentions: To date, five metabotropic S1P1–5 receptors for S1P have been identified which are members of the edg (endothelial differentiation gene) family of G-protein coupled receptors (GPCRs) [32]–[35]. To elucidate which S1P receptors were relevant for the regulation of thermal sensitivity in sensory neurons, we performed receptor mRNA expression profile analyses in DRG explants, in acutely dissociated neurons, and in primary DRG neuron enriched cultures. Quantitative PCR showed the following rank order of relative receptor mRNA expression for DRG explants: S1P3 > S1P1 > S1P2 > S1P4 > S1P5 (Fig. 3A, B). These explants contain neurons and non-neuronal cells including satellite cells, myelinating and non-myelinating Schwann cells, resident immune cells, endothelial cells and vascular immune cells [36]. S1P receptors have been found expressed in most of these cell types (for review see [37]. However, in preparations enriched with acutely isolated sensory neurons (<1% non-neuronal cells) and in 1-day-old neuron-enriched cultures mRNAs for S1P1, S1P2 and S1P3 but not S1P4 and S1P5 receptors were expressed. The relative expression levels of mRNA for S1P1–3 receptors were similar in acutely dissociated neurons and in 1-day-old cultures (Fig. 3A, B). Together this suggests that S1P1, S1P2 and S1P3 receptor subtypes were expressed in sensory neurons whilst S1P4 and S1P5 subtypes were not. We conclude, therefore, that in the explant preparations, mRNAs for S1P4 and S1P5 receptors were probably derived from non-neuronal cells within the DRG. Most nociceptive neurons are small to medium-sized peripherin-positive neurons [38]. They include glial-derived neurotrophic factor (GDNF)/protein typrosine kinase receptor (c-RET) dependent neurons with affinity of the isolectin B4 (I-B4+) and NGF/tyrosine kinase A (TrkA) dependent neurons that contain the neuropeptides calcitonin-gene related peptide (CGRP) and substance P [39]. Immunoreactivity for S1P1 receptor was found predominantly in small peripherin-IR neurons in DRG sections (Fig. 3C, D, E, F). In sections and neuron cultures, S1P1 immunoreactivity predominantly occurred in small I-B4+, Nf200-negative neurons. A smaller group of CGRP-IR neurons also displayed immunoreactivity for S1P1 (Table 1). Based on this co-expression profile, we predicted that the actions of S1P on nociceptor function were most likely mediated by the S1P1 receptor.

Bottom Line: Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue.We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors.Our data show that neuronally expressed S1P₁ receptors play a significant role in regulating nociceptor function and that S1P/S1P₁ signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

View Article: PubMed Central - PubMed

Affiliation: Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria. norbert.mair@i-med.ac.at

ABSTRACT
Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P₁ receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P₁ receptor. Our data show that neuronally expressed S1P₁ receptors play a significant role in regulating nociceptor function and that S1P/S1P₁ signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation.

Show MeSH
Related in: MedlinePlus