Differences in glutamate receptors and inflammatory cell numbers are associated with the resolution of pain in human rotator cuff tendinopathy

Benjamin John Floyd Dean, Sarah J B Snelling, Stephanie G Dakin, Richard J Murphy, Muhammad Kassim Javaid, Andrew Jonathan Carr, Benjamin John Floyd Dean, Sarah J B Snelling, Stephanie G Dakin, Richard J Murphy, Muhammad Kassim Javaid, Andrew Jonathan Carr

Abstract

Introduction: The relationship between peripheral tissue characteristics and pain symptoms in soft tissue inflammation is poorly understood. The primary aim of this study was to determine immunohistochemical differences in tissue obtained from patients with persistent pain and patients who had become pain-free after surgical treatment for rotator cuff tendinopathy. The secondary aim was to investigate whether there would be differences in glutaminergic and inflammatory gene expression between disease-derived and healthy control cells in vitro.

Methods: Supraspinatus tendon biopsies were obtained from nine patients with tendon pain before shoulder surgery and from nine further patients whose pain had resolved completely following shoulder surgery. Histological markers relating to the basic tendon characteristics, inflammation and glutaminergic signalling were quantified by immunohistochemical analysis. Gene expression of glutaminergic and inflammatory markers was determined in tenocyte explants derived from painful rotator cuff tendon tears in a separate cohort of patients and compared to that of explants from healthy control tendons. Dual labelling was performed to identify cell types expressing nociceptive neuromodulators.

Results: Tendon samples from patients with persistent pain demonstrated increased levels of metabotropic glutamate receptor 2 (mGluR2), kainate receptor 1 (KA1), protein gene product 9.5 (PGP9.5), CD206 (macrophage marker) and CD45 (pan-leucocyte marker) versus pain-free controls (p <0.05). NMDAR1 co-localised with CD206-positive cells, whereas PGP9.5 and glutamate were predominantly expressed by resident tendon cells. These results were validated by in vitro increases in the expression of mGluR2, N-methyl-D-aspartate receptor (NMDAR1), KA1, CD45, CD206 and tumour necrosis factor alpha (TNF-α) genes (p <0.05) in disease-derived versus control cells.

Conclusions: We conclude that differences in glutamate receptors and inflammatory cell numbers are associated with the resolution of shoulder pain in rotator cuff tendinopathy, and that disease-derived cells exhibit a distinctly different neuro-inflammatory gene expression profile to healthy control cells.

Figures

Fig. 1
Fig. 1
Basic histology was similar in the pain-free and painful groups. Photomicrographs show the haematoxylin and eosin staining in the rotator cuff tendon in the painful (a, b, c, d) and pain-free (e, f, g, h) groups. Scale bars: 40 μm
Fig. 2
Fig. 2
There were inflammatory differences between painful and pain-free rotator cuff tendons. Both the number of CD45 (pan-leucocyte marker) and CD206 (macrophage marker) positive cells were increased in the painful group versus the pain-free group. LDH expression was increased in the painful group, while VEGF was increased in the pain-free group. Photomicrographs depict the immunohistochemical staining of VEGF (a, e), LDH (b, f), CD45 (c, g), CD206 (d, h), in the painful and pain-free groups. Scale bars: 40 μm. LDH lactate dehydrogenase, VEGF vascular endothelial growth factor
Fig. 3
Fig. 3
There were glutaminergic differences between painful and pain-free rotator cuff tendons. The expression of several glutaminergic markers was different between groups; mGluR2 and KA1 were increased in the painful group, while mGluR7 was increased in the pain-free group. mGluR7 staining was exclusively of endothelial cells, while mGluR2 and KA1 staining was of both endothelial cells and resident tendon cells. PGP 9.5 expression (type B synoviocyte marker) was increased in the painful group. Photomicrographs depict the immunohistochemical staining of mGluR2 (a, e), mGluR7 (b, f), PGP 9.5 (c, g) and KA1 (d, h). Scale bars: 40 μm. KA1 kainate receptor 1, mGluR metabotropic glutamate receptor, PGP9.5 protein gene product 9.5
Fig. 4
Fig. 4
Representative immunofluorescent images of torn human supraspinatus to show cell types expressing nociceptive neuromodulators. Panel shows dual antibody labelling for CD206 (green) with (a) glutamate (violet), (b) PGP9.5 (violet) and (c) NMDAR1 (red). Cyan represents POPO-1 nuclear counterstain. NMDAR1 co-localised with CD206-positive cells, whereas PGP9.5 and glutamate were predominantly expressed by resident tendon cells. Scale bar: 20 μm. mGluR metabotropic glutamate receptor, NMDAR N-methyl-D-aspartate receptor, PGP9.5 protein gene product 9.5
Fig. 5
Fig. 5
Neuro-inflammatory genes are upregulated in cells derived from rotator cuff tendon tears versus control cells. The expression of the TNF-α (9.5-fold, p = 0.0009), CD45 (12.3-fold, p = 0.03), CD206 (2-fold, p = 0.004), NMDAR1 (3.5-fold, p = 0.003), KA1 (7.5-fold, p = 0.049) and mGluR2 (205-fold, p = 0.0006) genes was significantly increased in tendon tear-derived cells (a) proliferation, cell surface markers and matrix genes (b) cytokine and glutaminergic genes (n = 7 tendon tear-derived and 5 controls). The boxes represent median +/− interquartile range, while the whiskers represent range. Numbers in outlined boxes represent fold change in gene expression. Statistical significance denoted by *p <0.05, **p <0.01, ***p <0.001. KA1 kainate receptor 1, mGluR metabotropic glutamate receptor, NMDAR N-methyl-D-aspartate receptor, TNF-α tumour necrosis factor alpha

References

    1. Urwin M, Symmons D, Allison T, Brammah T, Busby H, Roxby M, et al. Estimating the burden of musculoskeletal disorders in the community: the comparative prevalence of symptoms at different anatomical sites, and the relation to social deprivation. Ann Rheum Dis. 1998;57:649–55. doi: 10.1136/ard.57.11.649.
    1. Judge A, Murphy RJ, Maxwell R, Arden NK, Carr AJ. Temporal trends and geographical variation in the use of subacromial decompression and rotator cuff repair of the shoulder in England. Bone Joint J. 2014;96-B:70–4. doi: 10.1302/0301-620X.96B1.32556.
    1. Chaudhury S, Gwilym SE, Moser J, Carr AJ. Surgical options for patients with shoulder pain. Nat Rev Rheumatol. 2010;6:217–26. doi: 10.1038/nrrheum.2010.25.
    1. Yamaguchi KDK, Middleton WD, Hildebolt CF, Galatz LM, Teefey SA. The demographic and morphological features of rotator cuff disease. A comparison of asymptomatic and symptomatic shoulders. J Bone Joint Surg Am. 2006;88:1699–704. doi: 10.2106/JBJS.E.00835.
    1. De Groote J, Ovreeide P, Mermuys K, Casselman J. Flexor tendon tenosynovitis with rice body formation in rheumatoid arthritis. JBR-BTR. 2014;97:123.
    1. Benazzo F, Zanon G, Klersy C, Marullo M. Open surgical treatment for chronic midportion Achilles tendinopathy: faster recovery with the soleus fibres transfer technique. Knee Surg Sports Traumatol Arthrosc. 2014
    1. Rees JD, Stride M, Scott A. Tendons - time to revisit inflammation. Br J Sports Med. 2014;48:1553–7. doi: 10.1136/bjsports-2012-091957.
    1. Franklin SL, Dean BJ, Wheway K, Watkins B, Javaid MK, Carr AJ. Up-regulation of glutamate in painful human supraspinatus tendon tears. Am J Sports Med. 2014;42:1955–62. doi: 10.1177/0363546514532754.
    1. Ackermann PW, Franklin SL, Dean BJ, Carr AJ, Salo PT, Hart DA. Neuronal pathways in tendon healing and tendinopathy--update. Front Biosci (Landmark Ed). 2014;19:1251–78. doi: 10.2741/4280.
    1. Schizas N, Weiss R, Lian O, Frihagen F, Bahr R, Ackermann PW. Glutamate receptors in tendinopathic patients. J Orthop Res. 2012;30:1447–52. doi: 10.1002/jor.22094.
    1. Newsholme P, Procopio J, Lima MM, Pithon-Curi TC, Curi R. Glutamine and glutamate--their central role in cell metabolism and function. Cell Biochem Funct. 2003;21:1–9. doi: 10.1002/cbf.1003.
    1. Dean BJ, Gwilym SE, Carr AJ. Why does my shoulder hurt? A review of the neuroanatomical and biochemical basis of shoulder pain. Br J Sports Med. 2013;47:1095–104. doi: 10.1136/bjsports-2012-091492.
    1. Dean BJ, Franklin SL, Carr AJ. A systematic review of the histological and molecular changes in rotator cuff disease. Bone Joint Res. 2012;1:158–66. doi: 10.1302/2046-3758.17.2000115.
    1. Murphy RJ, Daines MT, Carr AJ, Rees JL. An independent learning method for orthopaedic surgeons performing shoulder ultrasound to identify full-thickness tears of the rotator cuff. J Bone Joint Surg Am. 2013;95:266–72. doi: 10.2106/JBJS.K.00706.
    1. Dawson J, Fitzpatrick R, Carr A. Questionnaire on the perceptions of patients about shoulder surgery. J Bone Joint Surg. 1996;78:593–600.
    1. Murphy RJ, Floyd Dean BJ, Wheway K, Watkins B, Morrey ME, Carr AJ. A novel minimally invasive ultrasound-guided technique to biopsy supraspinatus tendon. Operat Tech Orthop. 2013;23:56–62. doi: 10.1053/j.oto.2013.05.003.
    1. Dean BJ, Franklin SL, Murphy RJ, Javaid MK, Carr AJ. Glucocorticoids induce specific ion-channel-mediated toxicity in human rotator cuff tendon: a mechanism underpinning the ultimately deleterious effect of steroid injection in tendinopathy? Br J Sports Med. 2014;48:1620–6. doi: 10.1136/bjsports-2013-093178.
    1. Cook JL, Feller JA, Bonar SF, Khan KM. Abnormal tenocyte morphology is more prevalent than collagen disruption in asymptomatic athletes’ patellar tendons. J Orthop Res. 2004;22:334–8. doi: 10.1016/j.orthres.2003.08.005.
    1. Poulsen RC, Carr AJ, Hulley PA. Protection against glucocorticoid-induced damage in human tenocytes by modulation of ERK, Akt, and forkhead signaling. Endocrinology. 2011;152:503–14. doi: 10.1210/en.2010-1087.
    1. Dean BJ, Franklin SL, Carr AJ. The peripheral neuronal phenotype is important in the pathogenesis of painful human tendinopathy: a systematic review. Clin Orthop Relat Res. 2013;471:3036–46. doi: 10.1007/s11999-013-3010-y.
    1. Tomita Y, Ozaki J, Sakurai G, Kondo T, Nakagaki K, Tamai S. Neurohistology of the subacromial bursa in rotator cuff tear. J Orthop Sci. 1997;2:295–300. doi: 10.1007/BF02488913.
    1. Gotoh M, Hamada K, Yamakawa H, Inoue A, Fukuda H. Increased substance P in subacromial bursa and shoulder pain in rotator cuff diseases. J Orthop Res. 1998;16:618–21. doi: 10.1002/jor.1100160515.
    1. Millar NL, Hueber AJ, Reilly JH, Xu Y, Fazzi UG, Murrell GA, et al. Inflammation is present in early human tendinopathy. Am J Sports Med. 2010;38:2085–91. doi: 10.1177/0363546510372613.
    1. Scott A, Lian O, Bahr R, Hart DA, Duronio V, Khan KM. Increased mast cell numbers in human patellar tendinosis: correlation with symptom duration and vascular hyperplasia. Br J Sports Med. 2008;42:753–7. doi: 10.1136/bjsm.2007.040212.
    1. Kragsnaes MS, Fredberg U, Stribolt K, Kjaer SG, Bendix K, Ellingsen T. Stereological quantification of immune-competent cells in baseline biopsy specimens from achilles tendons: results from patients with chronic tendinopathy followed for more than 4 years. Am J Sports Med. 2014;42:2435–45. doi: 10.1177/0363546514542329.
    1. Christian RA, Rossy WH, Sherman OH. Patellar tendinopathy - recent developments toward treatment. Bull Hosp Joint Dis (2013) 2014;72:217–24.
    1. Jeong DU, Lee CR, Lee JH, Pak J, Kang LW, Jeong BC, et al. Clinical applications of platelet-rich plasma in patellar tendinopathy. Biomed Res Int. 2014;2014:249498.
    1. Reilkoff RA, Bucala R, Herzog EL. Fibrocytes: emerging effector cells in chronic inflammation. Nat Rev Immunol. 2011;11:427–35. doi: 10.1038/nri2990.
    1. Iwanaga T, Shikichi M, Kitamura H, Yanase H, Nozawa-Inoue K. Morphology and functional roles of synoviocytes in the joint. Arch Histol Cytol. 2000;63:17–31. doi: 10.1679/aohc.63.17.
    1. Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 2010;233:233–55. doi: 10.1111/j.0105-2896.2009.00859.x.
    1. Julio-Pieper M, Flor PJ, Dinan TG, Cryan JF. Exciting times beyond the brain: metabotropic glutamate receptors in peripheral and non-neural tissues. Pharmacol Rev. 2011;63:35–58. doi: 10.1124/pr.110.004036.
    1. Madden DR. The structure and function of glutamate receptor ion channels. Nat Rev Neurosci. 2002;3:91–101. doi: 10.1038/nrn725.
    1. Alfredson H, Forsgren S, Thorsen K, Lorentzon R. In vivo microdialysis and immunohistochemical analyses of tendon tissue demonstrated high amounts of free glutamate and glutamate NMDAR1 receptors, but no signs of inflammation, in Jumper's knee. J Orthop Res. 2001;19:881–6. doi: 10.1016/S0736-0266(01)00016-X.
    1. Schizas N, Lian O, Frihagen F, Engebretsen L, Bahr R, Ackermann PW. Coexistence of up-regulated NMDA receptor 1 and glutamate on nerves, vessels and transformed tenocytes in tendinopathy. Scand J Med Sci Sports. 2010;20:208–15. doi: 10.1111/j.1600-0838.2009.00913.x.
    1. Zammataro M, Chiechio S, Montana MC, Traficante A, Copani A, Nicoletti F, et al. mGlu2 metabotropic glutamate receptors restrain inflammatory pain and mediate the analgesic activity of dual mGlu2/mGlu3 receptor agonists. Mol Pain. 2011;7:6. doi: 10.1186/1744-8069-7-6.
    1. Yamamoto T, Saito O, Aoe T, Bartolozzi A, Sarva J, Zhou J, et al. Local administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in peripheral pain in rats. Eur J Neurosci. 2007;25:147–58. doi: 10.1111/j.1460-9568.2006.05272.x.
    1. Gillard SE, Tzaferis J, Tsui HC, Kingston AE. Expression of metabotropic glutamate receptors in rat meningeal and brain microvasculature and choroid plexus. J Comp Neurol. 2003;461:317–32. doi: 10.1002/cne.10671.
    1. Sharp CD, Fowler M, Jackson TH, Houghton J, Warren A, Nanda A, et al. Human neuroepithelial cells express NMDA receptors. BMC Neurosci. 2003;4:28. doi: 10.1186/1471-2202-4-28.
    1. Papadia S, Hardingham GE. The dichotomy of NMDA receptor signalling. Neuroscientist. 2007;13:572–9. doi: 10.1177/1073858407305833.
    1. Flood S, Parri R, Williams A, Duance V, Mason D. Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors. Arthritis Rheum. 2007;56:2523–34. doi: 10.1002/art.22829.
    1. Piepoli T, Mennuni L, Zerbi S, Lanza M, Rovati LC, Caselli G. Glutamate signaling in chondrocytes and the potential involvement of NMDA receptors in cell proliferation and inflammatory gene expression. Osteoarthritis Cartilage. 2009;17:1076–83. doi: 10.1016/j.joca.2009.02.002.
    1. McNearney TA, Ma Y, Chen Y, Taglialatela G, Yin H, Zhang WR, et al. A peripheral neuroimmune link: glutamate agonists upregulate NMDA NR1 receptor mRNA and protein, vimentin, TNF-alpha, and RANTES in cultured human synoviocytes. Am J Physiol Regul Integr Comp Physiol. 2010;298:R584–98. doi: 10.1152/ajpregu.00452.2009.
    1. Bonnet CS, Williams AS, Gilbert SJ, Harvey AK, Evans BA, Mason DJ. AMPA/kainate glutamate receptors contribute to inflammation, degeneration and pain related behaviour in inflammatory stages of arthritis. Ann Rheum Dis. 2015;74:242–51. doi: 10.1136/annrheumdis-2013-203670.
    1. Spang C, Alfredson H, Ferguson M, Roos B, Bagge J, Forsgren S. The plantaris tendon in association with mid-portion Achilles tendinosis: tendinosis-like morphological features and presence of a non-neuronal cholinergic system. Histol Histopathol. 2013;28:623–32.
    1. Backman LJ, Andersson G, Fong G, Alfredson H, Scott A, Danielson P. Alpha-2 adrenergic stimulation triggers Achilles tenocyte hypercellularity: comparison between two model systems. Scand J Med Sci Sports. 2013;23:687–96. doi: 10.1111/j.1600-0838.2011.01442.x.
    1. Lakemeier S, Reichelt JJ, Patzer T, Fuchs-Winkelmann S, Paletta JR, Schofer MD. The association between retraction of the torn rotator cuff and increasing expression of hypoxia inducible factor 1alpha and vascular endothelial growth factor expression: an immunohistological study. BMC Musculoskelet Disord. 2010;11:230. doi: 10.1186/1471-2474-11-230.
    1. Lakemeier S, Braun J, Efe T, Foelsch C, Archontidou-Aprin E, Fuchs-Winkelmann S, et al. Expression of matrix metalloproteinases 1, 3, and 9 in differing extents of tendon retraction in the torn rotator cuff. Knee Surg Sports Traumatol Arthrosc. 2011;19:1760–5. doi: 10.1007/s00167-010-1367-y.
    1. Tillander B, Franzen L, Norlin R. Fibronectin, MMP-1 and histologic changes in rotator cuff disease. J Orthop Res. 2002;20:1358–64. doi: 10.1016/S0736-0266(02)00057-8.
    1. Tomonaga A, Hamada K, Gotoh M, Yamakawa H, Kobayashi K, Fukuda H. Expression of procollagen alpha 1 type III mRNA in rotator cuff tears. Tokai J Exp Clin Med. 2000;25:125–34.
    1. Kukkonen J, Joukainen A, Lehtinen J, Mattila KT, Tuominen EK, Kauko T, et al. Treatment of non-traumatic rotator cuff tears: a randomised controlled trial with one-year clinical results. Bone Joint J. 2014;96-B:75–81. doi: 10.1302/0301-620X.96B1.32168.
    1. Ketola S, Lehtinen J, Arnala I, Nissinen M, Westenius H, Sintonen H, et al. Does arthroscopic acromioplasty provide any additional value in the treatment of shoulder impingement syndrome?: a two-year randomised controlled trial. J Bone Joint Surg. 2009;91:1326–34. doi: 10.1302/0301-620X.91B10.22094.

Source: PubMed

스폰서 및 공동 작업자

건강 상태

약물 개입

3
구독하다