, Volume 26, Issue 3, pp 369–385 | Cite as

Metallic gold beads in hyaluronic acid: a novel form of gold-based immunosuppression? Investigations of the immunosuppressive effects of metallic gold on cultured J774 macrophages and on neuronal gene expression in experimental autoimmune encephalomyelitis

  • Dan Sonne PedersenEmail author
  • Thao Phuong Tran
  • Kamille Smidt
  • Bo Martin Bibby
  • Jørgen Rungby
  • Agnete Larsen


Multiple sclerosis (MS) is a neurodegenerative disease caused by recurring attacks of neuroinflammation leading to neuronal death. Immune-suppressing gold salts are used for treating connective tissue diseases; however, side effects occur from systemic spread of gold ions. This is limited by exploiting macrophage-induced liberation of gold ions (dissolucytosis) from gold surfaces. Injecting gold beads in hyaluronic acid (HA) as a vehicle into the cavities of the brain can delay clinical signs of disease progression in the MS model, experimental autoimmune encephalitis (EAE). This study investigates the anti-inflammatory properties of metallic gold/HA on the gene expression of tumor necrosis factor (Tnf-α), Interleukin (Il)-, Il-6, Il-10, Colony-stimulating factor (Csf)-v2, Metallothionein (Mt)-1/2, Bcl-2 associated X protein (Bax) and B cell lymphoma (Bcl)-2 in cultured J774 macrophages and in rodents with early stages of EAE. Cells grew for 5 days on gold/HA or HA, then receiving 1,000 ng/mL lipopolysaccharide (LPS) as inflammatory challenge. In the EAE experiment, 12 Lewis rats received gold injections and control groups included 11 untreated and 12 HA-treated EAE rats and five healthy animals. The experiment terminated day 9 when the first ten animals showed signs of EAE, only one of which were gold-treated (1p = 0.0367). Gene expression in the macrophages showed a statistically significant decrease in Il-6, Il- and Il-10-response to LPS; interestingly HA induced a statistically significant increase of Il-10. In the EAE model gene expression of inflammatory cytokines increased markedly. Compared to EAE controls levels of Tnf-α, Il-, Il-10, Il-6, IL-2, Ifn-γ, Il-17, transforming growth factor (Tgf)-β, superoxide dismutase (Sod)-2, Mt-2 and fibroblast growth factor (Fgf)-2 were lower in the gold-treated group. HA-treated animals expressed similar or intermediate levels. Omnibus testing for reduced inflammatory response following gold-treatment was not significant, but tendencies towards a decrease in the Sod-2, Fgf-2, Il- response and a higher Bdnf and IL-23 gene expression were seen. In conclusion, our findings support that bio-liberation of gold from metallic gold surfaces have anti-inflammatory properties similar to classic gold compounds, warranting further studies into the pharmacological potential of this novel gold-treatment and the possible synergistic effects of hyaluronic acid.


Anti-inflammation Dissolucytosis EAE Hyaluronic acid Macrophages Metallic gold 



The authors would like to thank Heidi Schou Knudsen and Karen Skjødt Sørensen for their excellent technical assistance and the animal department at The Department of Biomedicine for their help. We are grateful for financial support from the Aarhus University Research Foundation, Den Lægevidenskabelige Forskningsfond, Fonden af 17.12.1981 and Lykfeldts Legat.


  1. Agrawal SM, Yong VW (2007) Immunopathogenesis of multiple sclerosis. Int Rev Neurobiol 79:99–126CrossRefPubMedGoogle Scholar
  2. Al Qteishat A, Gaffney JJ, Krupinski J, Slevin M (2006) Hyaluronan expression following middle cerebral artery occlusion in the rat. Neuroreport 17:1111–1114CrossRefPubMedGoogle Scholar
  3. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303–1313CrossRefPubMedGoogle Scholar
  4. Back SA, Tuohy TM, Chen H, Wallingford N, Craig A, Struve J, Luo NL, Banine F, Liu Y, Chang A, Trapp BD, Bebo BFJ, Rao MS, Sherman LS (2005) Hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation. Nat Med 11:966–972PubMedGoogle Scholar
  5. Becher B, Segal BM (2011) T(H)17 cytokines in autoimmune neuro-inflammation. Curr Opin Immunol 23:707–712CrossRefPubMedGoogle Scholar
  6. Bosch X, Saiz A, Ramos-Casals M (2011) Monoclonal antibody therapy-associated neurological disorders. Nat Rev Neurol 7:165–172CrossRefPubMedGoogle Scholar
  7. Bot PT, Hoefer IE, Piek JJ, Pasterkamp G (2008) Hyaluronic acid: targeting immune modulatory components of the extracellular matrix in atherosclerosis. Curr Med Chem 15:786–791CrossRefPubMedGoogle Scholar
  8. Bovolenta R, Zucchini S, Paradiso B, Rodi D, Merigo F, Navarro Mora G, Osculati F, Berto E, Marconi P, Marzola A, Fabene PF, Simonato M (2010) Hippocampal FGF-2 and BDNF overexpression attenuates epileptogenesis-associated neuroinflammation and reduces spontaneous recurrent seizures. J Neuroinflammation 7:81CrossRefPubMedGoogle Scholar
  9. Brinkmann V, Billich A, Baumruker T, Heining P, Schmouder R, Francis G, Aradhye S, Burtin P (2010) Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat Rev Drug Discov 9:883–897CrossRefPubMedGoogle Scholar
  10. Campo GM, Avenoso A, Campo S, D’Ascola A, Nastasi G, Calatroni A (2010) Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes. Biochem Pharmacol 80:480–490CrossRefPubMedGoogle Scholar
  11. Campo GM, Avenoso A, D’Ascola A, Scuruchi M, Prestipino V, Calatroni A, Campo S (2012) Hyaluronan in part mediates IL-1beta-induced inflammation in mouse chondrocytes by up-regulating CD44 receptors. Gene 494:24–35CrossRefPubMedGoogle Scholar
  12. Cantor JO, Nadkarni PP (2006) Hyaluronan: the Jekyll and Hyde molecule. Inflamm Allergy Drug Targets 5:257–260CrossRefPubMedGoogle Scholar
  13. Cavaillon JM (1994) Cytokines and macrophages. Biomed Pharmacother 48:445–453CrossRefPubMedGoogle Scholar
  14. Chung RS, Hidalgo J, West AK (2008) New insight into the molecular pathways of metallothionein-mediated neuroprotection and regeneration. J Neurochem 104:14–20PubMedGoogle Scholar
  15. Constantinescu CS, Farooqi N, O’Brien K, Gran B (2011) Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol 164(4):1079–1106CrossRefPubMedGoogle Scholar
  16. Coyle P, Philcox JC, Carey LC, Rofe AM (2002) Metallothionein: the multipurpose protein. Cell Mol Life Sci 59:627–647CrossRefPubMedGoogle Scholar
  17. Danscher G (2002) In vivo liberation of gold ions from gold implants. Autometallographic tracing of gold in cells adjacent to metallic gold. Histochem Cell Biol 117:447–452CrossRefPubMedGoogle Scholar
  18. Danscher G, Larsen A (2010) Effects of dissolucytotic gold ions on recovering brain lesions. Histochem Cell Biol 133:367–373CrossRefPubMedGoogle Scholar
  19. Eisler R (2003) Chrysotherapy: a synoptic review. Inflamm Res 52:487–501CrossRefPubMedGoogle Scholar
  20. Frinchi M, Di Liberto V, Olivieri M, Fuxe K, Belluardo N, Mudo G (2010) FGF-2/FGFR1 neurotrophic system expression level and its basal activation do not account for the age-dependent decline of precursor cell proliferation in the subventricular zone of rat brain. Brain Res 1358:39–45CrossRefPubMedGoogle Scholar
  21. Gontiya G, Galgali SR (2012) Effect of hyaluronan on periodontitis: a clinical and histological study. J Indian Soc Periodontol 16:184–192CrossRefPubMedGoogle Scholar
  22. Haynes DR, Garrett IR, Whitehouse MW, Vernon-Roberts B (1988) Do gold drugs inhibit interleukin-1? Evidence from an in vitro lymphocyte activating factor assay. J Rheumatol 15:775–778PubMedGoogle Scholar
  23. Heldin P, Karousou E, Bernert B, Porsch H, Nishitsuka K, Skandalis SS (2008) Importance of hyaluronan-CD44 interactions in inflammation and tumorigenesis. Connect Tissue Res 49:215–218CrossRefPubMedGoogle Scholar
  24. Huang XN, Wang WZ, Fu J, Wang HB (2011) The relationship between aquaporin-4 expression and blood-brain and spinal cord barrier permeability following experimental autoimmune encephalomyelitis in the rat. Anat Rec (Hoboken) 294:46–54CrossRefGoogle Scholar
  25. Imrich H, Harzer K (2001) On the role of peripheral macrophages during active experimental allergic encephalomyelitis (EAE). J Neural Transm 108:379–395CrossRefPubMedGoogle Scholar
  26. Jiang D, Liang J, Noble PW (2011) Hyaluronan as an immune regulator in human diseases. Physiol Rev 91:221–264CrossRefPubMedGoogle Scholar
  27. Jovanovic DV, Di Battista JA, Martel-Pelletier J, Jolicoeur FC, He Y, Zhang M, Mineau F, Pelletier JP (1998) IL-17 stimulates the production and expression of proinflammatory cytokines, IL-beta and TNF-alpha, by human macrophages. J Immunol 160:3513–3521PubMedGoogle Scholar
  28. Kebir H, Ifergan I, Alvarez JI, Bernard M, Poirier J, Arbour N, Duquette P, Prat A (2009) Preferential recruitment of interferon-gamma-expressing TH17 cells in multiple sclerosis. Ann Neurol 66:390–402CrossRefPubMedGoogle Scholar
  29. Lampa J, Klareskog L, Ronnelid J (2002) Effects of gold on cytokine production in vitro; increase of monocyte dependent interleukin 10 production and decrease of interferon-gamma levels. J Rheumatol 29:21–28PubMedGoogle Scholar
  30. Larsen A, Stoltenberg M, Danscher G (2007) In vitro liberation of charged gold atoms: autometallographic tracing of gold ions released by macrophages grown on metallic gold surfaces. Histochem Cell Biol 128:1–6CrossRefPubMedGoogle Scholar
  31. Larsen A, Kolind K, Pedersen DS, Doering P, Pedersen MO, Danscher G, Penkowa M, Stoltenberg M (2008) Gold ions bio-released from metallic gold particles reduce inflammation and apoptosis and increase the regenerative responses in focal brain injury. Histochem Cell Biol 130(4):681–692CrossRefPubMedGoogle Scholar
  32. Locht LJ, Pedersen MO, Markholt S, Bibby BM, Larsen A, Penkowa M, Stoltenberg M, Rungby J (2010) Metallic silver fragments cause massive tissue loss in the mouse brain. Basic Clin Pharmacol Toxicol 109(1):1–10CrossRefGoogle Scholar
  33. Madhok R, Crilly A, Murphy E, Smith J, Watson J, Capell HA (1993) Gold therapy lowers serum interleukin 6 levels in rheumatoid arthritis. J Rheumatol 20:630–633PubMedGoogle Scholar
  34. Miles AT, Hawksworth GM, Beattie JH, Rodilla V (2000) Induction, regulation, degradation, and biological significance of mammalian metallothioneins. Crit Rev Biochem Mol Biol 35:35–70CrossRefPubMedGoogle Scholar
  35. Mix E, Meyer-Rienecker H, Hartung HP, Zettl UK (2010) Animal models of multiple sclerosis-Potentials and limitations. Prog Neurobiol 92(3):386–404CrossRefPubMedGoogle Scholar
  36. Miyamoto K, Nagaosa N, Motoyama M, Kataoka K, Kusunoki S (2009) Upregulation of water channel aquaporin-4 in experimental autoimmune encephalomyeritis. J Neurol Sci 276:103–107CrossRefPubMedGoogle Scholar
  37. Mudo G, Bonomo A, Di Liberto V, Frinchi M, Fuxe K, Belluardo N (2009) The FGF-2/FGFRs neurotrophic system promotes neurogenesis in the adult brain. J Neural Transm 116(8):995–1005CrossRefPubMedGoogle Scholar
  38. Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates. Academic Press, San DiegoGoogle Scholar
  39. Pedersen MO, Larsen A, Pedersen DS, Stoltenberg M, Penkova M (2009) Metallic gold treatment reduces proliferation of inflammatory cells, increases expression of VEGF and FGF, and stimulates cell proliferation in the subventricular zone following experimental traumatic brain injury. Histol Histopathol 24:573–586PubMedGoogle Scholar
  40. Pedersen MO, Larsen A, Stoltenberg M, Penkowa M (2010) Bio-released gold ions modulate expression of neuroprotective and hematopoietic factors after brain injury. Brain Res 1307:1–13CrossRefPubMedGoogle Scholar
  41. Pedersen DS, Fredericia PM, Pedersen MO, Stoltenberg M, Penkowa M, Danscher G, Rungby J, Larsen A (2012) Metallic gold slows disease progression, reduces cell death and induces astrogliosis while simultaneously increasing stem cell responses in an EAE rat model of multiple sclerosis. Histochem Cell Biol 138:787–802CrossRefPubMedGoogle Scholar
  42. Presta M, Andres G, Leali D, Dell’Era P, Ronca R (2009) Inflammatory cells and chemokines sustain FGF2-induced angiogenesis. Eur Cytokine Netw 20:39–50PubMedGoogle Scholar
  43. R Development Core Team (2011) R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. ISBN 3-900051-07-0.
  44. Rudick RA, Goelz SE (2011) Beta-interferon for multiple sclerosis. Exp Cell Res 317(9):1301–1311CrossRefPubMedGoogle Scholar
  45. Saidha S, Eckstein C, Calabresi PA (2012) New and emerging disease modifying therapies for multiple sclerosis. Ann N Y Acad Sci 1247:117–137CrossRefPubMedGoogle Scholar
  46. Schiff MH, Whelton A (2000) Renal toxicity associated with disease-modifying antirheumatic drugs used for the treatment of rheumatoid arthritis. Semin Arthritis Rheum 30:196–208CrossRefPubMedGoogle Scholar
  47. Schoenborn JR, Wilson CB (2007) Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol 96:41–101CrossRefPubMedGoogle Scholar
  48. Shao Y, Lu GL, Shen ZJ, He HC (2012) Reduction of intercellular adhesion molecule 1 may play a role in anti-inflammatory effect of hyaluronic acid in a rat model of severe non-bacterial cystitis. World J Urol (Epub ahead of print)Google Scholar
  49. Skundric DS (2005) Experimental models of relapsing-remitting multiple sclerosis: current concepts and perspective. Curr Neurovasc Res 2:349–362CrossRefPubMedGoogle Scholar
  50. Smidt K, Wogensen L, Brock B, Schmitz O, Rungby J (2006) Real-time PCR: housekeeping genes in the INS-1E beta-cell line. Horm Metab Res 38:8–11CrossRefPubMedGoogle Scholar
  51. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):Research0034CrossRefPubMedGoogle Scholar
  52. Vasak M, Meloni G (2011) Chemistry and biology of mammalian metallothioneins. J Biol Inorg Chem 16:1067–1078CrossRefPubMedGoogle Scholar
  53. Wang H, Zhan Y, Xu L, Feuerstein GZ, Wang X (2001) Use of suppression subtractive hybridization for differential gene expression in stroke: discovery of CD44 gene expression and localization in permanent focal stroke in rats. Stroke 32:1020–1027CrossRefPubMedGoogle Scholar
  54. Wang MJ, Kuo JS, Lee WW, Huang HY, Chen WF, Lin SZ (2006) Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages. J Neurochem 97:857–871CrossRefPubMedGoogle Scholar
  55. Yanni G, Nabil M, Farahat MR, Poston RN, Panayi GS (1994) Intramuscular gold decreases cytokine expression and macrophage numbers in the rheumatoid synovial membrane. Ann Rheum Dis 53:315–322CrossRefPubMedGoogle Scholar
  56. Zainali K, Danscher G, Jakobsen T, Jakobsen SS, Baas J, Moller P, Bechtold JE, Soballe K (2009) Effects of gold coating on experimental implant fixation. J Biomed Mater Res A 88:274–280PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Dan Sonne Pedersen
    • 1
    Email author
  • Thao Phuong Tran
    • 1
  • Kamille Smidt
    • 1
  • Bo Martin Bibby
    • 2
  • Jørgen Rungby
    • 1
  • Agnete Larsen
    • 1
  1. 1.Department of Biomedicine, PharmacologyAarhus UniversityAarhus CDenmark
  2. 2.Department of Public Health, Institute of BiostatisticsAarhus UniversityAarhus CDenmark

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