Inflammation Research

, Volume 65, Issue 7, pp 533–542 | Cite as

Hyaluronidase decreases neutrophils infiltration to the inflammatory site

  • Marcio Fronza
  • Cornélia Muhr
  • Denise Sayuri Calheiros da Silveira
  • Carlos Artério Sorgi
  • Stephen Fernandes de Paula Rodrigues
  • Sandra Helena Poliselli Farsky
  • Francisco Wanderley Garcia Paula-Silva
  • Irmgard Merfort
  • Lúcia Helena FaccioliEmail author
Original Research Paper



To evaluate the in vivo anti-inflammatory potential of bovine hyaluronidase (HYAL) using two different models of acute inflammation.


Air pouches were produced in the dorsal subcutaneous of mice and injected with phosphate saline solution or HYAL. The antiinflammatory action of HYAL was evaluated in carrageenan (Cg)-inflamed air pouches. After 4 and 24 h the cellular influx, protein exudation, cytokines and lipid mediators were evaluated. The action of HYAL on the rolling and adhesion of leukocytes was investigated in the LPS-stimulated mesenteric microcirculation by intravital microscopic.


Treatment with HYAL reduced the cellular influx and protein exudation in non-inflamed and inflamed air pouches. HYAL treatment of Cg-inflamed air pouch reduced the production of tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8), leukotriene B4 (LTB4) and LTC4, whereas prostaglandins E2 (PGE2) and D2 (PGD2) concentrations were unchanged. Histological analyses showed that HYAL administration diminished cell infiltration in the air-pouch lining. In LPS-stimulated mesenteric microcirculation, HYAL usage decreased rolling and adhesion of leukocytes, but did not affect the blood vessels diameters.


The results demonstrate that HYAL inhibited cellular recruitment, edema formation and pro-inflammatory mediators production, resulting in decreased adherence of leukocytes to blood vessels and tissue infiltration. Our data suggest that HYAL may be considered an effective candidate to ameliorate acute inflammation.


Air pouch Cytokines Lipid mediators Intravital microscopy Leukocyte–endothelial interactions Neutrophil influx Carrageenan LPS 



The authors are grateful to the São Paulo Research Foundation (FAPESP, Grants# 2011/23992-3; 2009/07169-5), to the German Academic Exchange Service (DAAD) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support. S.F. Rodrigues is a postdoctoral fellow from FAPESP (2011/02438-8).


  1. 1.
    El-Safory NS, Fazary AE, Lee CK. Hyaluronidases, a group of glycosidases: current and future perspectives. Carbohydr Polym. 2010;81:165–81.CrossRefGoogle Scholar
  2. 2.
    Girish KS, Kemparaju K. Inhibition of Naja naja venom hyaluronidase by plant-derived bioactive components and polysaccharides. Biochemistry (Mosc). 2005;70:948–52.CrossRefPubMedGoogle Scholar
  3. 3.
    Girish KS, Kemparaju K. The magic glue hyaluronan and its eraser hyaluronidase: a biological overview. Life Sci. 2007;80(21):1921–43.CrossRefPubMedGoogle Scholar
  4. 4.
    Fox JW. A brief review of the scientific history of several lesser-known snake venom proteins: l-amino acid oxidases, hyaluronidases and phosphodiesterases. Toxicon. 2013;62:75–82.CrossRefPubMedGoogle Scholar
  5. 5.
    Dunn AL, Heavner JE, Racz G, Day M. Hyaluronidase: a review of approved formulations, indications and off-label use in chronic pain management. Expert Opin Biol Ther. 2010;10:127–31.CrossRefPubMedGoogle Scholar
  6. 6.
    Kemparaju K, Girish KS. Snake venom hyaluronidase: a therapeutic target. Cell Biochem Funct. 2006;24(1):7–12.CrossRefPubMedGoogle Scholar
  7. 7.
    Ferguson EL, Roberts JL, Moseley R, Griffiths PC, Thomas DW. Evaluation of the physical and biological properties of hyaluronan and hyaluronan fragments. Int J Pharm. 2011;420(1):84–92.CrossRefPubMedGoogle Scholar
  8. 8.
    David-Raoudi M, Tranchepain F, Deschrevel B, Vincent JC, Bogdanowicz P, Boumediene K, Pujol JP. Differential effects of hyaluronan and its fragments on fibroblasts: relation to wound healing. Wound Repair Regen. 2008;16:274–87.CrossRefPubMedGoogle Scholar
  9. 9.
    Bitencourt CS, Pereira PA, Ramos SG, Sampaio SV, Arantes EC, Aronoff DM, Faccioli LH. Hyaluronidase recruits mesenchymal-like cells to the lung and ameliorates fibrosis. Fibrogenesis Tissue Repair. 2011. doi: 10.1186/1755-1536-4-3.PubMedPubMedCentralGoogle Scholar
  10. 10.
    Bourguignon LY, Wong G, Earle CA, Xia W. Interaction of low molecular weight hyaluronan with CD44 and toll-like receptors promotes the actin filament-associated protein 110-actin binding and MyD88-NFκB signaling leading to proinflammatory cytokine/chemokine production and breast tumor invasion. Cytoskeleton (Hoboken). 2011;68:671–93.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sidgwick GP, Iqbal SA, Bayat A. Altered expression of hyaluronan synthase and hyaluronidase mRNA may affect hyaluronic acid distribution in keloid disease compared with normal skin. Exp Dermatol. 2013;22(5):377–9.CrossRefPubMedGoogle Scholar
  12. 12.
    Fronza M, Caetano GF, Leite MN, Bitencourt CS, Paula-Silva FW, Andrade TA, Frade MA, Merfort I, Faccioli LH. Hyaluronidase modulates inflammatory response and accelerates the cutaneous wound healing. PLoS One. 2014. doi: 10.1371/journal.pone.0112297.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Huang Z, Zhao C, Chen Y, Cowell JA, Wei G, Kultti A, Huang L, Thompson CB, Rosengren S, Frost GI, Shepard HM. Recombinant human hyaluronidase PH20 does not stimulate an acute inflammatory response and inhibits lipopolysaccharide-induced neutrophil recruitment in the air pouch model of inflammation. J Immunol. 2014;192(11):5285–95.CrossRefPubMedGoogle Scholar
  14. 14.
    Sacca R, Cuff CA, Ruddle NH. Mediators of inflammation. Curr Opin Immunol. 1997;9(6):851–7.CrossRefPubMedGoogle Scholar
  15. 15.
    Rock KL, Latz E, Ontiveros F, Kono H. The sterile inflammatory response. Annu Rev Immunol. 2010;28:321–42.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chen GY, Nuñez G. Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol. 2010;10:826–37.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wright HL, Moots RJ, Bucknall RC, Edwards SW. Neutrophil function in inflammation and inflammatory diseases. Rheumatology (Oxford). 2010;49(9):1618–31.CrossRefGoogle Scholar
  18. 18.
    Kaplanski G, Marin V, Montero-Julian F, Mantovani A, Farnarier C. IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation. Trends Immunol. 2003;24(1):25–9.CrossRefPubMedGoogle Scholar
  19. 19.
    Faccioli LH, Nourshargh S, Moqbel R, Williams FM, Sehmi R, Kay AB, Williams TJ. The accumulation of 111In-eosinophils induced by inflammatory mediators, in vivo. Immunology. 1991;73(2):222–7.PubMedPubMedCentralGoogle Scholar
  20. 20.
    Medeiros AI, Silva CL, Malheiro A, Maffei CM, Faccioli LH. Leukotrienes are involved in leukocyte recruitment induced by live Histoplasma capsulatum or by the beta-glucan present in their cell wall. Br J Pharmacol. 1999;128(7):1529–37.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Flamand N, Mancuso P, Serezani CH, Brock TG. Leukotrienes: mediators that have been typecast as villains. Cell Mol Life Sci. 2007;64:2657–70.CrossRefPubMedGoogle Scholar
  22. 22.
    Henderson WR Jr. The role of leukotrienes in inflammation. Ann Intern Med. 1994;121(9):684–97.CrossRefPubMedGoogle Scholar
  23. 23.
    Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31(5):986–1000.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013;13(3):159–75.CrossRefPubMedGoogle Scholar
  25. 25.
    Aoki T, Narumiya S. Prostaglandins and chronic inflammation. Trends Pharmacol Sci. 2012;33:304–11.CrossRefPubMedGoogle Scholar
  26. 26.
    McDonald B, McAvoy EF, Lam F, Gill V, de la Motte C, Savani RC, Kubes P. Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids. J Exp Med. 2008;205(4):915–27.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Deutschman CS, Tracey KJ. Sepsis: current dogma and new perspectives. Immunity. 2014;40:463–75.CrossRefPubMedGoogle Scholar
  28. 28.
    Hack CE, Zeerleder S. The endothelium in sepsis: source of and a target for inflammation. Crit Care Med. 2001;29:S21–7.CrossRefPubMedGoogle Scholar
  29. 29.
    Duarte DB, Vasko MR, Fehrenbacher JC. Models of inflammation: carrageenan air pouch. Curr Protoc Pharmacol. 2012. doi: 10.1002/0471141755.PubMedCentralGoogle Scholar
  30. 30.
    Edwards JC, Sedgwick AD, Willoughby DA. The formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system. J Pathol. 1981;134(2):147–56.CrossRefPubMedGoogle Scholar
  31. 31.
    Rodrigues SF, Granger DN. Blood cells and endothelial barrier function. Tissue Barriers. 2015. doi: 10.4161/21688370.2014.978720.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Barioni ED, Santin JR, Machado ID, Rodrigues SF, Ferraz-de-Paula V, Wagner TM, Cogliati B, Corrêa Dos Santos M, Machado Mda S, de Andrade SF, Niero R, Farsky SH. Achyrocline satureioides (Lam.) D.C. hydroalcoholic extract inhibits neutrophil functions related to innate host defense. Evid Based Complement Alternat Med. 2013. doi: 10.1155/2013/787916.PubMedPubMedCentralGoogle Scholar
  33. 33.
    Verri WA Jr, Souto FO, Vieira SM, Almeida SC, Fukada SY, Xu D, Alves-Filho JC, Cunha TM, Guerrero AT, Mattos-Guimaraes RB, Oliveira FR, Teixeira MM, Silva JS, McInnes IB, Ferreira SH, Louzada-Junior P, Liew FY, Cunha FQ. IL-33 induces neutrophil migration in rheumatoid arthritis and is a target of anti-TNF therapy. Ann Rheum Dis. 2010;69(9):1697–703.CrossRefPubMedGoogle Scholar
  34. 34.
    Hwang SY, Kim JY, Kim KW, Park MK, Moon Y, Kim WU, Kim HY. IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-kappaB- and PI3-kinase/Akt-dependent pathways. Arthritis Res Ther. 2004;6(2):R120–8.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Troughton PR, Platt R, Bird H, el-Manzalawi E, Bassiouni M, Wright V. Synovial fluid interleukin-8 and neutrophil function in rheumatoid arthritis and seronegative polyarthritis. Br J Rheumatol. 1996;35(12):1244–51.CrossRefPubMedGoogle Scholar
  36. 36.
    Chen M, Lam BK, Kanaoka Y, Nigrovic PA, Audoly LP, Austen KF, Lee DM. Neutrophil-derived leukotriene B4 is required for inflammatory arthritis. J Exp Med. 2006;203:837–42.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Elmgreen J, Nielsen OH, Ahnfelt-Rønne I. Enhanced capacity for release of leucotriene B4 by neutrophils in rheumatoid arthritis. Ann Rheum Dis. 1987;46(7):501–5.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Mendes MT, Silveira PF. The interrelationship between leukotriene B4 and leukotriene-A4-hydrolase in collagen/adjuvant-induced arthritis in rats. Biomed Res Int. 2014. doi: 10.1155/2014/730421.Google Scholar
  39. 39.
    Tudan C, Jackson JK, Blanis L, Pelech SL, Burt HM. Inhibition of TNF-alpha-induced neutrophil apoptosis by crystals of calcium pyrophosphate dihydrate is mediated by the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways up-stream of caspase 3. J Immunol. 2000;165(10):5798–806.CrossRefPubMedGoogle Scholar
  40. 40.
    Adkison AM, Raptis SZ, Kelley DG, Pham CT. Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J Clin Invest. 2002;109:363–71.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Alten R, Gromnica-Ihle E, Pohl C, Emmerich J, Steffgen J, Roscher R, Sigmund R, Schmolke B, Steinmann G. Inhibition of leukotriene B4-induced CD11B/CD18 (Mac-1) expression by BIIL 284, a new long acting LTB4 receptor antagonist, in patients with rheumatoid arthritis. Ann Rheum Dis. 2004;63:170–6.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Crooks SW, Bayley DL, Hill SL, Stockley RA. Bronchial inflammation in acute bacterial exacerbations of chronic bronchitis: the role of leukotriene B4. Eur Respir J. 2000;15(2):274–80.CrossRefPubMedGoogle Scholar
  43. 43.
    Folco G, Rossoni G, Buccellati C, Berti F, Maclouf J, Sala A. Leukotrienes in cardiovascular diseases. Am J Respir Crit Care Med. 2000;161:S112–6.CrossRefPubMedGoogle Scholar
  44. 44.
    O’Byrne PM. Leukotrienes in the pathogenesis of asthma. Chest. 1997;111(2 Suppl):27S–34S.CrossRefPubMedGoogle Scholar
  45. 45.
    Steele VE, Holmes CA, Hawk ET, Kopelovich L, Lubet RA, Crowell JA, Sigman CC, Kelloff GJ. Lipoxygenase inhibitors as potential cancer chemopreventives. Cancer Epidemiol Biomarkers Prev. 1999;8(5):467–83.PubMedGoogle Scholar
  46. 46.
    Henry CB, Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan. Am J Physiol. 1999;277:H508–14.PubMedGoogle Scholar
  47. 47.
    Cabrales P, Vázquez BY, Tsai AG, Intaglietta M. Microvascular and capillary perfusion following glycocalyx degradation. J Appl Physiol. 1985;2007(102):2251–9.Google Scholar
  48. 48.
    Van den Berg BM, Vink H, Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ Res. 2003;92(6):592–4.CrossRefPubMedGoogle Scholar
  49. 49.
    Finsterbusch M, Voisin MB, Beyrau M, Williams TJ, Nourshargh S. Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF. J Exp Med. 2014;211:1307–14.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Johnsson C, Hällgren R, Elvin A, Gerdin B, Tufveson G. Hyaluronidase ameliorates rejection-induced edema. Transpl Int. 1999;12(4):235–43.CrossRefPubMedGoogle Scholar
  51. 51.
    Johnsson C, Hällgren R, Tufveson G. Hyaluronidase can be used to reduce interstitial edema in the presence of heparin. J Cardiovasc Pharmacol Ther. 2000;5(3):229–36.CrossRefPubMedGoogle Scholar
  52. 52.
    Yipp BG, Andonegui G, Howlett CJ, Robbins SM, Hartung T, Ho M, Kubes P. Profound differences in leukocyte-endothelial cell responses to lipopolysaccharide versus lipoteichoic acid. J Immunol. 2002;168(9):4650–8.CrossRefPubMedGoogle Scholar
  53. 53.
    Hakansson L, Venge P. The combined action of hyaluronic acid and fibronectin stimulates neutrophil migration. J Immunol. 1985;135(4):2735–9.PubMedGoogle Scholar
  54. 54.
    Khan AI, Kerfoot SM, Heit B, Liu L, Andonegui G, Ruffell B, Johnson P, Kubes P. Role of CD44 and hyaluronan in neutrophil recruitment. J Immunol. 2004;173(12):7594–601.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing 2016

Authors and Affiliations

  • Marcio Fronza
    • 1
    • 2
  • Cornélia Muhr
    • 3
  • Denise Sayuri Calheiros da Silveira
    • 1
  • Carlos Artério Sorgi
    • 1
  • Stephen Fernandes de Paula Rodrigues
    • 4
  • Sandra Helena Poliselli Farsky
    • 4
  • Francisco Wanderley Garcia Paula-Silva
    • 1
  • Irmgard Merfort
    • 3
  • Lúcia Helena Faccioli
    • 1
    Email author
  1. 1.Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão PretoUniversidade de São PauloRibeirão PretoBrazil
  2. 2.Departamento de FarmáciaUniversidade Vila VelhaVila VelhaBrazil
  3. 3.Department of Pharmaceutical Biology and BiotechnologyUniversity of FreiburgFreiburgGermany
  4. 4.Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical SciencesUniversity of São PauloSão PauloBrazil

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