Histochemistry and Cell Biology

, Volume 139, Issue 5, pp 691–715 | Cite as

Inflammatory markers CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil elastase in eccentric exercised human skeletal muscles

  • Gøran Paulsen
  • Ingrid Egner
  • Truls Raastad
  • Finn Reinholt
  • Simen Owe
  • Fredrik Lauritzen
  • Sverre-Henning Brorson
  • Satu Koskinen
Original Paper


The aim of the present study was to investigate leucocyte markers, CD11b, CD16, CD66b, CD68, myeloperoxidase and neutrophil elastase on skeletal muscle biopsies from biceps brachii after unaccustomed eccentric exercise followed by the second bout of exercise 3 weeks later. The subjects (10 subjects received COX-2 inhibitor (Celecoxib) and 13 subjects received placebo) were divided into three categories: mild, moderate and severe effect of eccentric exercise, according to the reduction and recovery of muscle force-generating capacity after performing 70 maximal eccentric actions with elbow flexors on an isokinetic dynamometer. The results showed that the CD66b antibody was applicable for localization of neutrophils in human skeletal muscle, whereas the other studied neutrophil markers recognized also other leucocytes than neutrophils. The number of CD66b positive cells in skeletal muscle was very low and was not affected by the exercise. The macrophage marker CD68 showed reactivity also against satellite cells and fibroblast-like cells in skeletal muscle and therefore cannot be applied as a quantitative value for inflammatory cells. Skeletal muscle fibre injury, shown as dystrophin negative fibres, was observed approximately in half of the biopsies at 4 and 7 days after the first exercise bout in the categories moderate and severe effect of eccentric exercise. These subjects represent the most prominent loss in muscle force-generating capacity both at the category and the individual levels. Furthermore, deformed skeletal muscle fibres were observed in five subjects in these categories after the second bout of exercise. The present results suggest that neutrophils are not involved in skeletal muscle fibre injury and the reduction in muscle force-generating capacity after a single bout of eccentric exercise is a good indirect indicator of muscle damage in humans. Furthermore, prolonged regeneration process could be one of the reasons for impaired peripheral muscle function after high-force eccentric exercise.


Human skeletal muscle Inflammatory cells Eccentric exercise Muscle damage Neutrophils Macrophages 



Bovine serum albumin


Cluster of differentiation 68


Celecoxib subject 01






Molecular weight


Optimal cutting temperature


Placebo subject 01


Region of interest

Supplementary material

418_2012_1061_MOESM1_ESM.doc (228 kb)
Supplementary material 1 (DOC 227 kb)
418_2012_1061_MOESM2_ESM.doc (274 kb)
Supplementary material 2 (DOC 273 kb)
418_2012_1061_MOESM3_ESM.pdf (2.1 mb)
Supplementary material 3 (PDF 2169 kb)


  1. Beaton LJ, Tarnopolsky MA, Phillips SM (2002) Contraction-induced muscle damage in humans following calcium channel blocker administration. J Physiol 544:849–859PubMedCrossRefGoogle Scholar
  2. Bröker BM, Edwards JC, Fanger MW, Lydyard PM (1990) The prevalence and distribution of macrophages bearing Fc gamma R I, Fc gamma R II, and Fc gamma R III in synovium. Scand J Rheumatol 19:123–135PubMedCrossRefGoogle Scholar
  3. Chen TC, Lin KY, Chen HL, Lin MJ, Nosaka K (2011) Comparison in eccentric exercise-induced muscle damage among four limb muscles. Eur J Appl Physiol 111:211–223PubMedCrossRefGoogle Scholar
  4. Clarkson PM, Hubal MJ (2002) Exercise-induced muscle damage in humans. Am J Phys Med Rehabil 81:S52–S69PubMedCrossRefGoogle Scholar
  5. Clarkson PM, Byrnes WC, Gillisson E, Harper E (1987) Adaptation to exercise-induced muscle damage. Clin Sci (Lond) 73:383–386Google Scholar
  6. Crameri RM, Langberg H, Teisner B, Magnusson P, Schrøder HD, Olesen JL, Jensen CH, Koskinen S, Suetta C, Kjaer M (2004) Enhanced procollagen processing in skeletal muscle after a single bout of eccentric loading in humans. Matrix Biol 23:259–264PubMedCrossRefGoogle Scholar
  7. Crameri RM, Aagaard P, Qvortrup K, Langberg H, Olesen J, Kjaer M (2007) Myofibre damage in human skeletal muscle: effects of electrical stimulation versus voluntary contraction. J Physiol 583:365–380PubMedCrossRefGoogle Scholar
  8. Evans WJ, Cannon JG (1991) The metabolic effects of exercise-induced muscle damage. Exerc Sport Sci Rev 19:99–125PubMedCrossRefGoogle Scholar
  9. Gottfried E, Kunz-Schughart LA, Weber A, Rehli M, Peuker A, Müller A, Kastenberger M, Brockhoff G, Andreesen R, Kreutz M (2008) Expression of CD68 in non-myeloid cell types. Scand J Immunol 67:453–463PubMedCrossRefGoogle Scholar
  10. Hellsten Y, Frandsen U, Orthenblad N, Sjødin B, Richter EA (1997) Xanthine oxidase in human skeletal muscle following eccentric exercise: a role in inflammation. J Physiol 498:239–248PubMedGoogle Scholar
  11. Hubal MJ, Rubinstein SR, Clarkson PM (2007) Mechanisms of variability in strength loss after muscle-lengthening actions. Med Sci Sports Exerc 39:461–468PubMedCrossRefGoogle Scholar
  12. Kawanaka N, Yamamura M, Aita T, Morita Y, Okamoto A, Kawashima M, Iwahashi M, Ueno A, Ohmoto Y, Makino H (2002) CD14+, CD16+ blood monocytes and joint inflammation in rheumatoid arthritis. Arthritis Rheum 46:2578–2586PubMedCrossRefGoogle Scholar
  13. Kunisch E, Fuhrmann R, Roth A, Winter R, Lungershausen W, Kinne RW (2004) Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry. Ann Rheum Dis 63:774–784PubMedCrossRefGoogle Scholar
  14. La Rocca G, Anzalone R, Farina F (2009) The expression of CD68 in human umbilical cord mesenchymal stem cells: new evidences of presence in non-myeloid cell types. Scand J Immunol 70:161–162PubMedCrossRefGoogle Scholar
  15. Lauritzen F, Paulsen G, Raastad T, Bergersen LH, Owe SG (2009) Gross ultrastructural changes and necrotic fiber segments in elbow flexor muscles after maximal voluntary eccentric action in humans. J Appl Physiol 107:1923–1934PubMedCrossRefGoogle Scholar
  16. MacIntyre DL, Reid WD, McKenzie DC (1995) Delayed muscle soreness. The inflammatory response to muscle injury and its clinical implications. Sports Med 20:24–40PubMedCrossRefGoogle Scholar
  17. MacNeil LG, Baker SK, Stevic I, Tarnopolsky MA (2011) 17β-estradiol attenuates exercise-induced neutrophil infiltration in men. Am J Physiol Regul Integr Comp Physiol 300:R1443–R1451PubMedCrossRefGoogle Scholar
  18. Mahoney DJ, Safdar A, Parise G, Melov S, Fu M, MacNeil L, Kaczor J, Payne ET, Tarnopolsky MA (2008) Gene expression profiling in human skeletal muscle during recovery from eccentric exercise. Am J Physiol Regul Integr Comp Physiol 294:R1901–R1910PubMedCrossRefGoogle Scholar
  19. Malm C, Nyberg P, Engstrom M, Sjodin B, Lenkei R, Ekblom B, Lundberg I (2000) Immunological changes in human skeletal muscle and blood after eccentric exercise and multiple biopsies. J Physiol 529:243–262PubMedCrossRefGoogle Scholar
  20. Malm C, Sjödin TL, Sjöberg B, Lenkei R, Renström P, Lundberg IE, Ekblom B (2004) Leukocytes, cytokines, growth factors and hormones in human skeletal muscle and blood after uphill or downhill running. J Physiol 556:983–1000PubMedCrossRefGoogle Scholar
  21. McHugh MP (2003) Recent advances in the understanding of the repeated bout effect: the protective effect against muscle damage from a single bout of eccentric exercise. Scand J Med Sci Sports 13:88–97PubMedCrossRefGoogle Scholar
  22. Middleton SJ, Li D, Wharton S, Reynolds PD, Wraight EP, Hunter JO (1995) Validation of 99Tcm-HMPAO leucocyte scintigraphy in ulcerative colitis by comparison with histology. Br J Radiol 68:1061–1066PubMedCrossRefGoogle Scholar
  23. Mikkelsen UR, Langberg H, Helmark IC, Skovgaard D, Andersen LL, Kjaer M, Mackey AL (2009) Local NSAID infusion inhibits satellite cell proliferation in human skeletal muscle after eccentric exercise. J Appl Physiol 107:1600–1611PubMedCrossRefGoogle Scholar
  24. Nosaka K, Sakamoto K (2001) Effect of elbow joint angle on the magnitude of muscle damage to the elbow flexors. Med Sci Sports Exerc 33:22–29PubMedGoogle Scholar
  25. Paulsen G, Egner IM, Drange M, Langberg H, Benestad HB, Fjeld JG, Hallén J, Raastad T (2010a) A COX-2 inhibitor reduces muscle soreness, but does not influence recovery and adaptation after eccentric exercise. Scand J Med Sci Sports 20:e195–e207PubMedCrossRefGoogle Scholar
  26. Paulsen G, Crameri R, Benestad HB, Fjeld JG, Mørkrid L, Hallén J, Raastad T (2010b) Time course of leukocyte accumulation in human muscle after eccentric exercise. Med Sci Sports Exerc 42:75–85PubMedGoogle Scholar
  27. Peake J, Nosaka K, Suzuki K (2005) Characterization of inflammatory responses to eccentric exercise in humans. Exerc Immunol Rev 11:64–85PubMedGoogle Scholar
  28. Peterson JM, Trappe TA, Mylona E, White F, Lambert CP, Evans WJ, Pizza FX (2003) Ibuprofen and acetaminophen: effect on muscle inflammation after eccentric exercise. Med Sci Sports Exerc 35:892–896PubMedCrossRefGoogle Scholar
  29. Prior BM, Jayaraman RC, Reid RW, Cooper TG, Foley JM, Dudley GA, Meyer RA (2001) Biarticular and monoarticular muscle activation and injury in human quadriceps muscle. Eur J Appl Physiol 85:185–190PubMedCrossRefGoogle Scholar
  30. Przybyla B, Gurley C, Harvey JF, Bearden E, Kortebein P, Evans WJ, Sullivan DH, Peterson CA, Dennis RA (2006) Aging alters macrophage properties in human skeletal muscle both at rest and in response to acute resistance exercise. Exp Gerontol 41:320–327PubMedCrossRefGoogle Scholar
  31. Raastad T, Risoy BA, Benestad HB, Fjeld JG, Hallen J (2003) Temporal relation between leukocyte accumulation in muscles and halted recovery 10–20 h after strength exercise. J Appl Physiol 95:2503–2509PubMedGoogle Scholar
  32. Sayers SP, Clarkson PM (2001) Force recovery after eccentric exercise in males and females. Eur J Appl Physiol 84:122–126PubMedCrossRefGoogle Scholar
  33. Scott A, Khan KM, Cook JL, Duronio V (2004) What is “inflammation”? Are we ready to move beyond Celsus? Br J Sports Med 38:248–249PubMedCrossRefGoogle Scholar
  34. Smith LL (1991) Acute inflammation: the underlying mechanism in delayed onset muscle soreness? Med Sci Sports Exerc 23:542–551PubMedGoogle Scholar
  35. Stauber WT, Clarkson PM, Fritz VK, Evans WJ (1990) Extracellular matrix disruption and pain after eccentric muscle action. J Appl Physiol 69:868–874PubMedGoogle Scholar
  36. Stupka N, Tarnopolsky MA, Yardley NJ, Phillips SM (2001) Cellular adaptation to repeated eccentric exercise-induced muscle damage. J Appl Physiol 91:1669–1678PubMedGoogle Scholar
  37. Warren GL, Ingalls CP, Shah SJ, Armstrong RB (1999) Uncoupling of in vivo torque production from EMG in mouse muscles injured by eccentric contractions. J Physiol 515:609–619PubMedCrossRefGoogle Scholar
  38. Ziegler-Heitbrock L (2007) The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 81:584–592PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Gøran Paulsen
    • 1
  • Ingrid Egner
    • 2
  • Truls Raastad
    • 1
  • Finn Reinholt
    • 3
  • Simen Owe
    • 4
  • Fredrik Lauritzen
    • 4
  • Sverre-Henning Brorson
    • 3
  • Satu Koskinen
    • 3
  1. 1.Department of Physical PerformanceNorwegian School of Sport ScienceOsloNorway
  2. 2.Department of Molecular BiosciencesUniversity of OsloOsloNorway
  3. 3.Department of PathologyUniversity of Oslo and Oslo University HospitalOsloNorway
  4. 4.Department of Anatomy and Centre for Molecular Biology and NeuroscienceUniversity of OsloOsloNorway

Personalised recommendations