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Comparative Study
. 2011 Jul;113(1):175-83.
doi: 10.1213/ANE.0b013e31821a0258. Epub 2011 May 19.

Neuropeptides contribute to peripheral nociceptive sensitization by regulating interleukin-1β production in keratinocytes

Xiaoyou Shi  1 , Liping Wang, Xiangqi Li, Peyman Sahbaie, Wade S Kingery, J David Clark
Affiliations
Comparative Study

Neuropeptides contribute to peripheral nociceptive sensitization by regulating interleukin-1β production in keratinocytes

Xiaoyou Shi et al. Anesth Analg. 2011 Jul.

Abstract

Background: It is increasingly evident that there is a close connection between the generation of cutaneous inflammatory cytokines and elevated neuropeptide signaling in complex regional pain syndrome (CRPS) patients. Previously, we observed in the rat tibia fracture model of CRPS that activation of caspase-1 containing NALP1 inflammasomes was required for interleukin (IL)-1β production in keratinocytes, and that administration of an IL-1 receptor antagonist (anakinra) reduced the fracture-induced hindpaw mechanical allodynia. We therefore hypothesized that neuropeptides lead to nociceptive sensitization through activation of the skin's innate immune system by enhancing inflammasome expression and caspase-1 activity.

Methods: We determined whether the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) require IL-1β to support nociceptive sensitization when injected into mouse hindpaw skin by testing mechanical allodynia. We then investigated whether these neuropeptides could stimulate production of IL-1β in a keratinocyte cell line (REKs), and could increase the expression of inflammasome component proteins including NALP1 and caspase-1. Finally, we determined whether neuropeptide-stimulated IL-1β production required activation of caspase-1 and cathepsin B.

Results: Intraplantar injections of SP and CGRP lead to allodynia in mouse hindpaws but CGRP was approximately 10-fold less potent in causing this response. Moreover, systemic administration of the IL-1 receptor (IL-1R) antagonist anakinra prevented sensitization after neuropeptide injection. Also, mouse skin keratinocytes express IL-1R, which is up-regulated after local neuropeptide application. In vitro data demonstrated that both SP and CGRP increased IL-1β gene and protein expression in REKs in a dose-dependent manner. Furthermore, SP time- and dose-dependently up-regulated NALP1 and caspase-1 mRNA and protein levels in REKs. In contrast, CGRP time- and dose-dependently enhanced NALP1 and caspase-1 mRNA levels without causing a significant change in NALP1 or caspase-1 protein expression in REKs. Inhibition of caspase-1 activity using the selective inhibitor Ac-YVAD-CHO reduced SP and, less effectively, CGRP induced increases in IL-1β production in REK cells. The selective cathepsin B inhibitor CA-74Me inhibited neuropeptide induced IL-1β production in REKs as well.

Conclusions: Collectively, these results demonstrate that neuropeptides induce nociceptive sensitization by enhancing IL-1 β production in keratinocytes. Neuropeptides rely on both caspase-1 and cathepsin B for this enhanced production. Neurocutaneous signaling involving neuropeptide activation of the innate immunity may contribute to pain in CRPS patients.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Interleukin (IL)-1 β receptor antagonist (IL-1ra, anakinra, 120 mg/kg, i.p.) prevents the mechanical sensitization induced by intraplantar injection of various doses of substance P (SP) or calcitonin-gene related peptide (CGRP). A) SP (300 ng and 1000 ng) caused significant mechanical allodynia lasting between 1 and 6 hours; whereas the anakinra preinjected mice displayed reduced hindpaw mechanical allodynia caused by SP. B) CGRP (3 μg and 10 μg) caused significant mechanical allodynia between 1 and 2 hours; whereas the anakinra preinjected mice displayed reduced hindpaw mechanical allodynia induced by CGRP. Values are means ± SE. N=6 mice for all experimental groups. * p<0.05, ** p<0.01, *** p<0.001 for differences relative to baseline; # p<0.05 for differences between the non-anakinra and anakinra pretreated mice that were injected with the same dose of SP or CGRP.
Figure 2
Figure 2
Neuropeptide mediated up-regulation of epidermal interleukin (IL)-1 receptor (IL-1R) expression. A) Immunohistochemistry of IL-1R and keratin before and after (2h) Substance P (SP) injection of hindpaw skin. B) The IL-1 receptor mRNA expression in the hindpaw skin 2 hours after administration of SP 1000 ng/15 μl and vehicle.
Figure 2
Figure 2
Neuropeptide mediated up-regulation of epidermal interleukin (IL)-1 receptor (IL-1R) expression. A) Immunohistochemistry of IL-1R and keratin before and after (2h) Substance P (SP) injection of hindpaw skin. B) The IL-1 receptor mRNA expression in the hindpaw skin 2 hours after administration of SP 1000 ng/15 μl and vehicle.
Figure 3
Figure 3
Neuropeptides Substance P (SP) and calcitonin-gene related peptide (CGRP) time- and dose-dependently increased the interleukin (IL)-1β mRNA level as measured by real-time polymerase chain reaction (PCR) and protein level as measured by ELISA (Fig. 2A, B, C, and D). Values are means ± SE. * p<0.05, ** p<0.01, *** p<0.001 vs. the vehicle-treated control cells (C).
Figure 4
Figure 4
Neuropeptides induced gene and protein expression of inflammasome component, NALP1 in the REK cultures. A) Substance P (SP) stimulated NAPL1 gene expression in a dose-dependent manner at 3 and 24 h posttreatment, respectively. B) Western blot band appearances (upper panel) and scanned densities presented as a bar graph (lower panel) for the full length NAPL1 and the short form NALP1 (NALP1s) in cultures treated with and without SP. C) calcitonin-gene related peptide (CGRP) dose-dependently stimulated NAPL1 gene expression at 3 and 24 h posttreatment, separately. D) Western blot band appearances (upper panel) and scanned densities presented as a bar graph (lower panel) for the full length NAPL1 and NALP1s in cultures treated with and without CGRP. Values are means ± SE. * p<0.05, ** p<0.01, *** p<0.001 vs. the vehicle-treated control cells (C).
Figure 5
Figure 5
Neuropeptides induced gene and protein expression of inflammasome component, caspase-1 in REK cells. A) Substance P (SP) stimulated caspase-1 gene expression in a dose-dependent manner at 3 and 24 h post-treatment, respectively. B) Western blot band appearances (upper panel) and scanned densities presented as a bar graph (lower panel) for the full length and the cleaved caspase-1 in cultures treated with and without SP. C) calcitonin-gene related peptide (CGRP) dose-dependently stimulated caspase-1 gene expression was mainly observed at 3h posttreatment. D) Western blot band appearances (upper panel) and scanned densities presented as a bar graph (lower panel) for the full length and the cleaved caspase-1 for cultures treated with and without CGRP. Values are means ± SE. * p<0.05, ** p<0.01, *** p<0.001 vs. the vehicle-treated control cells (C).
Figure 6
Figure 6
Caspase-1 inhibitor, Ac-YVAD-CHO inhibited the Substance P (SP) (10−8 M), not calcitonin-gene related peptide (CGRP) (10−8 M), induced interleukin (IL)-1β protein production (A and B) in the REK cultures. Values are means ± SE. ** p<0.01 vs. the control cells. # p<0.05 vs. the cultures treated with SP or CGRP.
Figure 7
Figure 7
Cathepsin B inhibitor, CA-74Me dose-dependently inhibited the Substance P (SP) (10−8 M) and calcitonin-gene related peptide (CGRP) (10−8 M) induced interleukin (IL)-1β protein production (A and B) in the REK cultures, respectively. Values are means ± SE. * p<0.05, ** p<0.01 vs. the vehicle-treated control cells.
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References

    1. Birklein F, Schmelz M. Neuropeptides, neurogenic inflammation and complex regional pain syndrome (CRPS) Neurosci Lett. 2008;437:199–202. - PubMed
    1. McDougall JJ. Arthritis and pain. Neurogenic origin of joint pain. Arthritis Res Ther. 2006;8:220. - PMC - PubMed
    1. Sahbaie P, Shi X, Guo TZ, Qiao Y, Yeomans DC, Kingery WS, Clark JD. Role of substance P signaling in enhanced nociceptive sensitization and local cytokine production after incision. Pain. 2009;145:341–9. - PMC - PubMed
    1. Huygen FJ, De Bruijn AG, De Bruin MT, Groeneweg JG, Klein J, Zijlstra FJ. Evidence for local inflammation in complex regional pain syndrome type 1. Mediators Inflamm. 2002;11:47–51. - PMC - PubMed
    1. Schinkel C, Gaertner A, Zaspel J, Zedler S, Faist E, Schuermann M. Inflammatory mediators are altered in the acute phase of posttraumatic complex regional pain syndrome. Clin J Pain. 2006;22:235–9. - PubMed

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