Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell–derived leukocyte elastase

Abstract

Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy1,2,3,4, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve-injured groups, which can be difficult to validate, particularly with respect to the specificity of pain modulation5. To circumvent this, we used two outbred lines of rats6, which are genetically similar except for being genetically segregated as a result of selective breeding for differences in neuropathic pain hypersensitivity7. SerpinA3N, a serine protease inhibitor, was upregulated in the dorsal root ganglia (DRG) after nerve injury, which was further validated for its mouse homolog. Mice lacking SerpinA3N developed more neuropathic mechanical allodynia than wild-type (WT) mice, and exogenous delivery of SerpinA3N attenuated mechanical allodynia in WT mice. T lymphocytes infiltrate the DRG after nerve injury and release leukocyte elastase (LE), which was inhibited by SerpinA3N derived from DRG neurons. Genetic loss of LE or exogenous application of a LE inhibitor (Sivelastat) in WT mice attenuated neuropathic mechanical allodynia. Overall, we reveal a novel and clinically relevant role for a member of the serpin superfamily and a leukocyte elastase and crosstalk between neurons and T cells in the modulation of neuropathic pain.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Serpina3n is upregulated in rats with low neuropathic pain sensitivity.
Figure 2: Serpina3n is upregulated in mouse lumbar DRGs after spared nerve injury, a model of neuropathic pain.
Figure 3: SerpinA3N attenuates mechanical allodynia after nerve injury.
Figure 4: Leukocyte elastase (LE) is a substrate for SerpinA3N, and it promotes neuropathic allodynia.

Similar content being viewed by others

References

  1. Costigan, M. et al. Multiple chronic pain states are associated with a common amino acid-changing allele in KCNS1. Brain 133, 2519–2527 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Nissenbaum, J. et al. Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2. Genome Res. 20, 1180–1190 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sorge, R.E. et al. Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity. Nat. Med. 18, 595–599 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Tegeder, I. et al. GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence. Nat. Med. 12, 1269–1277 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Persson, A.K. et al. Correlational analysis for identifying genes whose regulation contributes to chronic neuropathic pain. Mol. Pain 5, 7 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Devor, M. & Raber, P. Heritability of symptoms in an experimental model of neuropathic pain. Pain 42, 51–67 (1990).

    Article  CAS  PubMed  Google Scholar 

  7. Ziv-Sefer, S., Raber, P., Barbash, S. & Devor, M. Unity vs. diversity of neuropathic pain mechanisms: allodynia and hyperalgesia in rats selected for heritable predisposition to spontaneous pain. Pain 146, 148–157 (2009).

    Article  PubMed  Google Scholar 

  8. Seijffers, R., Mills, C.D. & Woolf, C.J. ATF3 increases the intrinsic growth state of DRG neurons to enhance peripheral nerve regeneration. J. Neurosci. 27, 7911–7920 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Horvath, A.J. et al. The murine orthologue of human antichymotrypsin: a structural paradigm for clade A3 serpins. J. Biol. Chem. 280, 43168–43178 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Sipione, S. et al. Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells. J. Immunol. 177, 5051–5058 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Gettins, P.G. Serpin structure, mechanism, and function. Chem. Rev. 102, 4751–4804 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Lee, W.L. & Downey, G.P. Leukocyte elastase: physiological functions and role in acute lung injury. Am. J. Respir. Crit. Care Med. 164, 896–904 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Pham, C.T. Neutrophil serine proteases: specific regulators of inflammation. Nat. Rev. Immunol. 6, 541–550 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. García, P.S., Gulati, A. & Levy, J.H. The role of thrombin and protease-activated receptors in pain mechanisms. Thromb. Haemost. 103, 1145–1151 (2010).

    Article  PubMed  Google Scholar 

  15. Kawasaki, Y. et al. Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. Nat. Med. 14, 331–336 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ferry, G. et al. Activation of MMP-9 by neutrophil elastase in an in vivo model of acute lung injury. FEBS Lett. 402, 111–115 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. Jackson, P.L. et al. Human neutrophil elastase-mediated cleavage sites of MMP-9 and TIMP-1: implications to cystic fibrosis proteolytic dysfunction. Mol. Med. 16, 159–166 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kawabata, K. et al. ONO-5046, a novel inhibitor of human neutrophil elastase. Biochem. Biophys. Res. Commun. 177, 814–820 (1991).

    Article  CAS  PubMed  Google Scholar 

  19. McMahon, S.B. & Malcangio, M. Current challenges in glia-pain biology. Neuron 64, 46–54 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Scholz, J. & Woolf, C.J. The neuropathic pain triad: neurons, immune cells and glia. Nat. Neurosci. 10, 1361–1368 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Hu, P., Bembrick, A.L., Keay, K.A. & McLachlan, E.M. Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve. Brain Behav. Immun. 21, 599–616 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Kim, C.F. & Moalem-Taylor, G. Detailed characterization of neuro-immune responses following neuropathic injury in mice. Brain Res. 1405, 95–108 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Gehrig, S., Mall, M.A. & Schultz, C. Spatially resolved monitoring of neutrophil elastase activity with ratiometric fluorescent reporters. Angew. Chem. Int. Edn Engl. 51, 6258–6261 (2012).

    Article  CAS  Google Scholar 

  24. Costigan, M. et al. T-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain-like hypersensitivity. J. Neurosci. 29, 14415–14422 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Irving, J.A., Pike, R.N., Lesk, A.M. & Whisstock, J.C. Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res. 10, 1845–1864 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Irving, J.A. et al. Serpins in prokaryotes. Mol. Biol. Evol. 19, 1881–1890 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Alam, S.R., Newby, D.E. & Henriksen, P.A. Role of the endogenous elastase inhibitor, elafin, in cardiovascular injury: from epithelium to endothelium. Biochem. Pharmacol. 83, 695–704 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Reardon, C. et al. Thymic stromal lymphopoetin-induced expression of the endogenous inhibitory enzyme SLPI mediates recovery from colonic inflammation. Immunity 35, 223–235 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Belaaouaj, A. et al. Mice lacking neutrophil elastase reveal impaired host defense against Gram-negative bacterial sepsis. Nat. Med. 4, 615–618 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Griffin, R.S. et al. Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity. J. Neurosci. 27, 8699–8708 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bolstad, B.M., Irizarry, R.A., Astrand, M. & Speed, T.P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).

    Article  PubMed  Google Scholar 

  33. Gentleman, R.C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Díaz, E. et al. Molecular analysis of gene expression in the developing pontocerebellar projection system. Neuron 36, 417–434 (2002).

    Article  PubMed  Google Scholar 

  35. Decosterd, I. & Woolf, C.J. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 87, 149–158 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Luo, C. et al. Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity. PLoS Biol. 10, e1001283 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Costigan, M. et al. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci. 3, 16 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Blackshaw, S. & Snyder, S.H. Parapinopsin, a novel catfish opsin localized to the parapineal organ, defines a new gene family. J. Neurosci. 17, 8083–8092 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Montmayeur, J.P., Liberles, S.D., Matsunami, H. & Buck, L.B. A candidate taste receptor gene near a sweet taste locus. Nat. Neurosci. 4, 492–498 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Schaeper, U. et al. Distinct requirements for Gab1 in Met and EGF receptor signaling in vivo. Proc. Natl. Acad. Sci. USA 104, 15376–15381 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lu, J. et al. Pain in experimental autoimmune encephalitis: a comparative study between different mouse models. J. Neuroinflammation 9, 233 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Basso, A.S. et al. Reversal of axonal loss and disability in a mouse model of progressive multiple sclerosis. J. Clin. Invest. 118, 1532–1543 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kim, C.F. & Moalem-Taylor, G. Detailed characterization of neuro-immune responses following neuropathic injury in mice. Brain Res. 1405, 95–108 (2011).

    Article  CAS  PubMed  Google Scholar 

  44. Jiang, P. et al. hESC-derived Olig2+ progenitors generate a subtype of astroglia with protective effects against ischaemic brain injury. Nat. Commun. 4, 2196 (2013).

    Article  PubMed  CAS  Google Scholar 

  45. Gangadharan, V. et al. A novel biological role for the phospholipid lysophosphatidylinositol in nociceptive sensitization via activation of diverse G protein signalling pathways in sensory nerves in vivo. Pain 154, 2801–2812 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Xu, Q. et al. In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery. PLoS ONE 7, e32581 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Wang, Z. et al. Differentiation of neuronal cells from NIH/3T3 fibroblasts under defined conditions. Dev. Growth Differ. 53, 357–365 (2011).

    Article  PubMed  Google Scholar 

  48. Hirota, Y. et al. Roles of planar cell polarity signaling in maturation of neuronal precursor cells in the postnatal mouse olfactory bulb. Stem Cells 30, 1726–1733 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Chiang, H.S. et al. GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses. Nat. Immunol. 15, 63–71 (2014).

    Article  CAS  PubMed  Google Scholar 

  50. Allman, D., Li, J. & Hardy, R.R. Commitment to the B lymphoid lineage occurs before DH-JH recombination. J. Exp. Med. 189, 735–740 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ko, S.Y. et al. alpha-Galactosylceramide can act as a nasal vaccine adjuvant inducing protective immune responses against viral infection and tumor. J. Immunol. 175, 3309–3317 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Charles, N., Hardwick, D., Daugas, E., Illei, G.G. & Rivera, J. Basophils and the T helper 2 environment can promote the development of lupus nephritis. Nat. Med. 16, 701–707 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Andoniou, C.E. et al. Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity. Nat. Immunol. 6, 1011–1019 (2005).

    Article  CAS  PubMed  Google Scholar 

  54. Papatriantafyllou, M. et al. Dickkopf-3, an immune modulator in peripheral CD8 T-cell tolerance. Proc. Natl. Acad. Sci. USA 109, 1631–1636 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Gehrig, S., Mall, M.A. & Schultz, C. Spatially resolved monitoring of neutrophil elastase activity with ratiometric fluorescent reporters. Angew. Chem. Int. Edn Engl. 51, 6258–6261 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors are grateful to R. LeFaucheur for secretarial assistance and to D. Baumgartl-Ahlert for technical assistance. We acknowledge support from the Interdisciplinary Neurobehavioral Core, Heidelberg, for the behavioral experiments performed here. We are grateful to M. Meister for his help in the analysis of FACS data. This work was supported by a European Research Council (ERC) Advanced Investigator Grant (PAINPLASTICITY; project no. 294293) to R.K., by grants from the Deutsche Forschungsgemeinschaft (DFG; MA 2081/4-1) to M.A.M., by grants from the Deutsche Forschungsgemeinschaft (SFB 938) to B.A., by grants from the US National Institutes of Health (5 R01 NS038253 and 2R37NS039518 to C.W., and R01 NS074430 to M.C.), and by a grant from the Israel Science Foundation to M.D. M.A.M. and R.K. are members of the Molecular Medicine Partnership Unit, Heidelberg. R.K. is a principal investigator in the Excellence Cluster 'CellNetworks' of Heidelberg University. M.A.M. is a member of the German Center for Lung Research. L.V. was partially supported by a PhD fellowship from CellNetworks and by the Hartmut-Hoffmann Berling International Graduate School for Cellular and Molecular Biology. M.S. was partially supported by a post-doctoral fellowship from CellNetworks. A.L. was partially supported by the RO1DE022912 grant from the US National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

L.V., D.E.S., A.L., K.K.B., M.S., D.H., S.P., P.R., R.S.G., C.N., S.G. and M.C. performed experiments and analyzed data; R.K., M.C., C.J.W., S.D.L., M.D., B.A., M.A.M. designed and supervised experiments; R.K., L.V. and M.C. primarily wrote the manuscript.

Corresponding author

Correspondence to Rohini Kuner.

Ethics declarations

Competing interests

The authors have a patent application pending based on the data herein on the use of leukocyte elastase inhibitors against pain of neuropathic origin (patent application number: EP14200012.4 at the European Patent Office).

Supplementary information

Supplementary Text and Figures

Supplementary Notes 1 and 2, Supplementary Table legends 1 and 2 and Supplementary Figures 1–12 (PDF 5934 kb)

Supplementary Table 1 (XLS 14140 kb)

Supplementary Table 2 (XLS 31 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vicuña, L., Strochlic, D., Latremoliere, A. et al. The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell–derived leukocyte elastase. Nat Med 21, 518–523 (2015). https://doi.org/10.1038/nm.3852

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3852

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing