Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 374, Issue 4, pp 265–273 | Cite as

Neutrophils-derived peroxynitrite contributes to acute hyperalgesia and cell influx in zymosan arthritis

  • Mirna M. Bezerra
  • Susan D. Brain
  • Virgínia C. C. Girão
  • Stan Greenacre
  • Julie Keeble
  • Francisco A. C. Rocha
Original Article


We investigated the contribution of neutrophils to joint hyperalgesia and peroxynitrite formation in zymosan arthritis. Rats received 1 mg zymosan intra-articular, and joint hyperalgesia was measured using the rat knee-joint articular incapacitation test. After 6 h, joint exudates were collected by aspiration for the assessment of cell influx, myeloperoxidase activity, and nitrite (as an index of nitric oxide formation) levels. Nitrotyrosine content, used as an index of peroxynitrite formation, was measured in joint exudates, using enzyme-linked immunosorbent assay. A group of rats was rendered neutropenic through the administration of a rabbit anti-rat neutrophil antibody (2 ml kg−1, i.p.) 30 min before injection of 1 mg zymosan intra-articular. Other groups received uric acid (100 or 250 mg kg−1, i.p.), the peroxynitrite scavenger, 30 min before 1 mg zymosan intra-articular. Controls received the vehicle. The significant inhibition of joint hyperalgesia in neutropenic animals was associated to significantly decreased cell influx, myeloperoxidase activity, nitric oxide, and nitrotyrosine levels in the joint exudates, as compared to naive rats. Uric acid administration inhibited both hyperalgesia and cell influx, as compared to controls. Neutrophils are involved in both nitric oxide and peroxynitrite formation in zymosan arthritis, thereby contributing to acute joint hyperalgesia. Scavenging of reactive nitrogen species (e.g. peroxynitrite) inhibits neutrophil migration and joint hyperalgesia in the acute phase of zymosan arthritis in rats.


Inflammation Peroxynitrite Neutrophils Zymosan Uric Acid 



disease modifying anti-rheumatic drugs



N-nitro-l-arginine methyl ester


leukotriene B4




nitric oxide


nitric oxide synthase




paw elevation time




rheumatoid arthritis




superoxide dismutase





The authors thank Fundação Cearense de Desenvolvimento Científico e Tecnológico (FUNCAP) and CNPq, Brazil.


  1. Baldus S, Eiserich JP, Mani A, Castro L, Figueroa M, Chumley P, Ma W, Tousson A, White CR, Bullard DC, Brennan ML, Lusis AJ, Moore KP, Freeman BA (2001) Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration. J Clin Invest 108:1759–1770PubMedCrossRefGoogle Scholar
  2. Beckman JS, Ye YZ, Anderson PG, Chen J, Accavitti MA, Tarpey MM, White CR (1994) Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe-Seyler 375:81–88Google Scholar
  3. Bezerra MM, Brain SD, Greenacre S, Jerônimo SM, de Melo LB, Keeble J, da Rocha FA (2004) Reactive nitrogen species scavenging, rather than nitric oxide inhibition, protects from articular cartilage damage in rat zymosan-induced arthritis. Br J Pharmacol 141:172–182PubMedCrossRefGoogle Scholar
  4. Boughton-Smith NK, Ghelani A (1995) Role of induced nitric oxide synthase and increased NO levels in zymosan peritonitis in the rat. Inflamm Res 44:S149–S150PubMedCrossRefGoogle Scholar
  5. Boulos C, Jiang H, Balazy M (2000) Diffusion of peroxynitrite into the human platelet inhibits cyclooxygenase via nitration of tyrosine residues. J Pharmacol Exp Ther 293:222–229PubMedGoogle Scholar
  6. Brennan ML, Wu W, Fu X, Shen Z, Song W, Frost H, Vadseth C, Narine L, Lenkiewicz E, Borchers MT, Lusis AJ, Lee JJ, Lee NA, Abu-Soud HM, Ischiropoulos H, Hazen SL (2002) A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species. J Biol Chem 277:17415–17427PubMedCrossRefGoogle Scholar
  7. Crooks SW, Stockley RA (1998) Leukotriene B4. Int J Biochem Cell Biol 30:173–178PubMedCrossRefGoogle Scholar
  8. da Rocha FA, de Brum-Fernandes AJ (2002) Evidence that peroxynitrite affects human osteoblast proliferation and differentiation. J Bone Miner Res 17:434–442PubMedCrossRefGoogle Scholar
  9. Eiserich JP, Cross CE, Jones AD, Halliwell B, van der Vliet A (1996) Formation of nitrating and chlorinating species by reaction of nitrite with hypochlorous acid—a novel mechanism for nitric oxide-mediated protein modification. J Biol Chem 271:19199–19208PubMedCrossRefGoogle Scholar
  10. Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B, van der Vliet A (1998) Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391:393–397PubMedCrossRefGoogle Scholar
  11. Feldmann M, Brennan FM, Maini RN (1996) Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14:397–440PubMedCrossRefGoogle Scholar
  12. Ferreira SH, Duarte ID, Lorenzetti BB (1991) The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via nitric oxide release. Eur J Pharmacol 201:121–122PubMedCrossRefGoogle Scholar
  13. Greenacre SA, Rocha FA, Rawlingson A, Meinerikandathevan S, Poston RN, Ruiz E, Halliwell B, Brain SD (2002) Protein nitration in cutaneous inflammation in the rat: essential role of inducible nitric oxide synthase and polymorphonuclear leukocytes. Br J Pharmacol 136:985–994PubMedCrossRefGoogle Scholar
  14. Halliwell B (1982) Production of superoxide, hydrogen peroxide and hydroxyl radicals by phagocytic cells: a cause of chronic inflammatory disease? Cell Biol Int Rep 6:529–542PubMedCrossRefGoogle Scholar
  15. Hampton MB, Kettle AJ, Winterbourn CC (1998) Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92:3007–3017PubMedGoogle Scholar
  16. Harris ED, Budd RC, Firestein GS, Genovese MC, Sergent JS, Ruddy S, Sledge CB (2004) Kelley’s textbook of rheumatology, 7th edn. Saunders, PhiladelphiaGoogle Scholar
  17. Hazen SL, Zhang R, Shen Z, Wu W, Podrez EA, MacPherson JC, Schmitt D, Mitra SN, Mukhopadhyay C, Chen Y, Cohen PA, Hoff HF, Abu-Soud HM (1999) Formation of nitric oxide-derived oxidants by myeloperoxidase in monocytes: pathways for monocyte-mediated protein nitration and lipid peroxidation in vivo. Circ Res 85:950–958PubMedGoogle Scholar
  18. Holthusen H, Arndt JO (1994) Nitric oxide evokes pain in humans on intracutaneous injection. Neurosci Lett 165:71–74PubMedCrossRefGoogle Scholar
  19. Ischiropoulos H, Zhu L, Beckman JS (1992) Peroxynitrite formation from macrophage-derived nitric oxide. Arch Biochem Biophys 298:446–451PubMedCrossRefGoogle Scholar
  20. Kaur H, Halliwell B (1994) Evidence for nitric oxide-mediated oxidative damage in chronic inflammation—nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett 350:9–12PubMedCrossRefGoogle Scholar
  21. Khalil Z, Liu T, Helme RD (1999) Free radicals contribute to the reduction in peripheral vascular responses and the maintenance of thermal hyperalgesia in rats with chronic constriction injury. Pain 79:31–37PubMedCrossRefGoogle Scholar
  22. Khan J, Brennand DM, Bradley N, Gao B, Bruckdorfer R, Jacobs M (1998) 3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method. Biochem J 330:795–801PubMedGoogle Scholar
  23. Kim HK, Park SK, Zhou JL, Taglialatela G, Chung K, Coggeshall RE, Chung JM (2004) Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain. Pain 111:116–124PubMedCrossRefGoogle Scholar
  24. Lawand NB, Willis WD, Westlund KN (1997) Blockade of joint inflammation and secondary hyperalgesia by l-NAME, a nitric oxide synthase inhibitor. NeuroReport 8:895–899PubMedCrossRefGoogle Scholar
  25. Liu T, Knight KR, Tracey DJ (2000) Hyperalgesia due to nerve injury-role of peroxynitrite. Neuroscience 97:125–131PubMedCrossRefGoogle Scholar
  26. Ma G, Al-Shabrawey M, Johnson JA, Datar R, Tawfik HE, Guo D, Caldwell RB, Caldwell RW (2006) Protection against myocardial ischemia/reperfusion injury by short-term diabetes: enhancement of VEGF formation, capillary density, and activation of cell survival signaling. Naunyn Schmiedeberg’s Arch Pharmacol 373:415–427CrossRefGoogle Scholar
  27. O’Dell JR (2004) Therapeutic strategies for rheumatoid arthritis. N Engl J Med 350:2591–2602PubMedCrossRefGoogle Scholar
  28. Ofulue AF, Ko M (1999) Effects of depletion of neutrophils or macrophages on development of cigarette smoke-induced emphysema. Am J Physiol 277:L97–L105PubMedGoogle Scholar
  29. Peters RR, Krepsky PB, Siqueira-Júnior JM, Rocha JCS, Bezerra MM, Ribeiro RA, Brum-Fernandes AJ, Farias MR, Rocha FAC, Ribeiro-do-Valle RM (2003) Nitric oxide and cyclooxygenase may participate in the analgesic fraction from Wilbrandia ebracteata. Life Sci 73:2185–2197PubMedCrossRefGoogle Scholar
  30. Rocha FA, Aragão AG Jr, Oliveira RC, Pompeu MM, Vale MR, Ribeiro RA (1999) Periarthritis promotes gait disturbance in zymosan-induced arthritis in rats. Inflamm Res 48:485–490PubMedCrossRefGoogle Scholar
  31. Rocha JCS, Peixoto MEB, Jancar S, Cunha FQ, Ribeiro RA, Rocha FAC (2002) Dual effect of nitric oxide in articular inflammatory pain in zymosan-induced arthritis in rats. Br J Pharmacol 136:588–596PubMedCrossRefGoogle Scholar
  32. Rocha FA, Teixeira MM, Rocha JC, Girão VC, Bezerra MM, Ribeiro RA, Cunha FQ (2004) Blockade of leukotriene B4 prevents articular incapacitation in rat zymosan-induced arthritis. Eur J Pharmacol 497:81–86PubMedCrossRefGoogle Scholar
  33. Salvemini D, Manning PT, Zweifel BS, Seibert K, Connor J, Currie MG, Needleman P, Masferrer JL (1995) Dual inhibition of nitric oxide and prostaglandin production contributes to the anti-inflammatory properties of nitric oxide synthase inhibitors. J Clin Invest 96:301–308PubMedCrossRefGoogle Scholar
  34. Salvemini D, Wang ZQ, Bourdon DM, Stern MK, Currie MG, Manning PT (1996) Evidence of peroxynitrite involvement in the carrageenan-induced rat paw edema. Eur J Pharmacol 303:217–220PubMedCrossRefGoogle Scholar
  35. Sato E, Simpson KL, Grisham MB, Koyama S, Robbins RA (2000) Reactive nitrogen and oxygen species attenuate interleukin-8-induced neutrophil chemotactic activity in vitro. J Biol Chem 275:10826–10830PubMedCrossRefGoogle Scholar
  36. Sousa AM, Prado WA (2001) The dual effect of a nitric oxide donor in nociception. Brain Res 897:9–19PubMedCrossRefGoogle Scholar
  37. Squadrito GL, Pryor WA (1995) The formation of peroxynitrite in vivo from nitric oxide and superoxide. Chem Biol Interact 96:203–206PubMedCrossRefGoogle Scholar
  38. Tonussi CR, Ferreira SH (1992) Rat-knee joint incapacitation test: an objective screen for central and peripheral analgesics. Pain 48:421–427PubMedCrossRefGoogle Scholar
  39. Wagner R, Heckman HM, Myers RR (1998) Wallerian degeneration and hyperalgesia after peripheral nerve injury are glutathione-dependent. Pain 77:173–179PubMedCrossRefGoogle Scholar
  40. Wang ZQ, Porreca F, Cuzzocrea S, Galen K, Lightfoot R, Masini E, Muscoli C, Mollace V, Ndengele M, Ischiropoulos H, Salvemini D (2004) A newly identified role for superoxide in inflammatory pain. J Pharmacol Exp Ther 309:869–878PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Mirna M. Bezerra
    • 1
  • Susan D. Brain
    • 2
  • Virgínia C. C. Girão
    • 1
  • Stan Greenacre
    • 2
  • Julie Keeble
    • 2
  • Francisco A. C. Rocha
    • 3
    • 4
  1. 1.Department of Physiology and Pharmacology, Faculty of Medicine of SobralFederal University of CearáSobralBrazil
  2. 2.Centre for Cardiovascular Biology and Medicine, New Hunt’s HouseKing’s College LondonLondonUK
  3. 3.Department of Internal MedicineFederal University of CearáFortalezaBrazil
  4. 4.Faculty of MedicineFederal University of CearáFortalezaBrazil

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