Skip to main content

Advertisement

SpringerLink
Log in

Bilirubin-induced immunostimulant effects and toxicity vary with neural cell type and maturation state

  • Original Paper
  • Published:
Acta Neuropathologica Aims and scope Submit manuscript

Abstract

Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the neonatal period. The increased vulnerability of premature infants to unconjugated bilirubin (UCB)-induced brain damage may be due to a proneness of immature nerve cells to UCB-toxic stimulus. Thus, in this study, we evaluated UCB-induced cell death, glutamate release and cytokine production, in astrocytes and neurons cultured for different days, in order to relate the differentiation state with cell vulnerability to UCB. The age-dependent activation of the nuclear factor-κB (NF-κB), an important transcription factor involved in inflammation, was also investigated. Furthermore, responsiveness of neurons and astrocytes to UCB were compared in order to identify the most susceptible to each induced effect, as an approach to what happens in vivo. The results clearly showed that immature nerve cells are more vulnerable than the most differentiated ones to UCB-induced cell death, glutamate release and tumour necrosis factor (TNF)-α secretion. Moreover, astrocytes seem to be more competent cells in releasing glutamate and in producing an inflammatory response when injured by UCB. Activation of NF-κB by UCB also presents a cell-age-dependent pattern, and values vary with neural cell type. Again, astrocytes have the highest activation levels, which are correlated with the greater amount of cytokine production observed in these cells. These results contribute to a better knowledge of the mechanisms leading to UCB encephalopathy by elucidation of age- and type-related differences in neural cell responses to UCB.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Abney ER, Bartlett PP, Raff MC (1981) Astrocytes, ependymal cells, and oligodendrocytes develop on schedule in dissociated cell cultures of embryonic rat brain. Dev Biol 83:301–310

    Article  PubMed  CAS  Google Scholar 

  2. American Academy of Pediatrics (2004) Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 114:297–316

    Article  PubMed  Google Scholar 

  3. Amit Y, Brenner T (1993) Age-dependent sensitivity of cultured rat glial cells to bilirubin toxicity. Exp Neurol 121:248–255

    Article  PubMed  CAS  Google Scholar 

  4. Bakalkin GY, Yakovleva T, Terenius L (1993) NF-κB-like factors in the murine brain. Developmentally-regulated and tissue-specific expression. Brain Res Mol Brain Res 20:137–146

    Article  PubMed  CAS  Google Scholar 

  5. Baldwin AS Jr (1996) The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol 14:649–683

    Article  PubMed  CAS  Google Scholar 

  6. Bales KR, Du Y, Dodel RC, Yan GM, Hamilton-Byrd E, Paul SM (1998) The NF-κB/Rel family of proteins mediates Aβ-induced neurotoxicity and glial activation. Brain Res Mol Brain Res 57:63–72

    Article  PubMed  CAS  Google Scholar 

  7. Barna BP, Estes ML, Jacobs BS, Hudson S, Ransohoff RM (1990) Human astrocytes proliferate in response to tumor necrosis factor α. J Neuroimmunol 30:239–243

    Article  PubMed  CAS  Google Scholar 

  8. Blondeau JP, Beslin A, Chantoux F, Francon J (1993) Triiodothyronine is a high-affinity inhibitor of amino acid transport system L1 in cultured astrocytes. J Neurochem 60:1407–1413

    Article  PubMed  CAS  Google Scholar 

  9. Breder CD, Tsujimoto M, Terano Y, Scott DW, Saper CB (1993) Distribution and characterization of tumor necrosis factor-alpha-like immunoreactivity in the murine central nervous system. J Comp Neurol 337:543–567

    Article  PubMed  CAS  Google Scholar 

  10. Brewer GJ (1997) Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 71:143–155

    Article  PubMed  CAS  Google Scholar 

  11. Brito MA, Rosa AI, Fernandes A, Falcão AS, Silva RFM, Brites D (2005) Hyperbilirubinemia induces oxidative stress in rat brain primary neuronal cultures. Free Radic Res 39(Suppl. 1):S52

    Google Scholar 

  12. Cai Z, Pan ZL, Pang Y, Evans OB, Rhodes PG (2000) Cytokine induction in fetal rat brains and brain injury in neonatal rats after maternal lipopolysaccharide administration. Pediatr Res 47:64–72

    Article  PubMed  CAS  Google Scholar 

  13. Cai Z, Pang Y, Lin S, Rhodes PG (2003) Differential roles of tumor necrosis factor-α and interleukin-1β in lipopolysaccharide-induced brain injury in the neonatal rat. Brain Res 975:37–47

    Article  PubMed  CAS  Google Scholar 

  14. Cauley K, Verma IM (1994) κB enhancer-binding complexes that do not contain NF-κB are developmentally regulated in mammalian brain. Proc Natl Acad Sci USA 91:390–394

    Article  PubMed  CAS  Google Scholar 

  15. Da SJ, Pierrat B, Mary JL, Lesslauer W (1997) Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes. J Biol Chem 272:28373–28380

    Article  PubMed  Google Scholar 

  16. Dalman C, Cullberg J (1999) Neonatal hyperbilirubinaemia—a vulnerability factor for mental disorder? Acta Psychiatr Scand 100:469–471

    Article  PubMed  CAS  Google Scholar 

  17. Dennery PA, Seidman DS, Stevenson DK (2001) Neonatal hyperbilirubinemia. N Engl J Med 344:581–590

    Article  PubMed  CAS  Google Scholar 

  18. Falcão AS, Fernandes A, Brito MA, Silva RFM, Brites D (2005) Bilirubin-induced inflammatory response, glutamate release, and cell death in rat cortical astrocytes are enhanced in younger cells. Neurobiol Dis 20:199–206

    Article  PubMed  CAS  Google Scholar 

  19. Fernandes A, Silva RFM, Falcão AS, Brito MA, Brites D (2004) Cytokine production, glutamate release and cell death in rat cultured astrocytes treated with unconjugated bilirubin and LPS. J Neuroimmunol 153:64–75

    Article  PubMed  CAS  Google Scholar 

  20. Fernandes A, Falcão AS, Silva RFM, Gordo AC, Gama MJ, Brito MA, Brites D (2006) Inflammatory signaling pathways involved in astroglial activation by unconjugated bilirubin. J Neurochem 96:1667–1679

    Article  PubMed  CAS  Google Scholar 

  21. Gadient RA, Otten U (1994) Expression of interleukin-6 (IL-6) and interleukin-6 receptor (IL-6R) mRNAs in rat brain during postnatal development. Brain Res 637:10–14

    Article  PubMed  CAS  Google Scholar 

  22. Gadient RA, Cron KC, Otten U (1990) Interleukin-1β and tumor necrosis factor-α synergistically stimulate nerve growth factor (NGF) release from cultured rat astrocytes. Neurosci Lett 117:335–340

    Article  PubMed  CAS  Google Scholar 

  23. Gendron RL, Nestel FP, Lapp WS, Baines MG (1991) Expression of tumor necrosis factor-α in the developing nervous system. Int J Neurosci 60:129–136

    Article  PubMed  CAS  Google Scholar 

  24. Gilmore JH, Jarskog LF, Vadlamudi S, Lauder JM (2004) Prenatal infection and risk for schizophrenia: IL-1β, IL-6 and TNF-α inhibit cortical neuron dendrite development. Neuropsychopharmacology 29:1221–1229

    Article  PubMed  CAS  Google Scholar 

  25. Grima G, Benz B, Parpura V, Cuenod M, Do KQ (2003) Dopamine-induced oxidative stress in neurons with glutathione deficit: implication for schizophrenia. Schizophr Res 62:213–224

    Article  PubMed  Google Scholar 

  26. Gourley GR (1997) Bilirubin metabolism and kernicterus. Adv Pediatr 44:173–229

    PubMed  CAS  Google Scholar 

  27. Grojean S, Koziel V, Vert P, Daval JL (2000) Bilirubin induces apoptosis via activation of NMDA receptors in developing rat brain neurons. Exp Neurol 166:334–341

    Article  PubMed  CAS  Google Scholar 

  28. Hanisch UK (2002) Microglia as a source and target of cytokines. Glia 40:140–155

    Article  PubMed  Google Scholar 

  29. Hankø E, Hansen TWR, Almaas R, Lindstad J, Rootwelt T (2005) Bilirubin induces apoptosis and necrosis in human NT2-N neurons. Pediatr Res 57:179–184

    Article  PubMed  CAS  Google Scholar 

  30. Hansen TWR (1995) Acute entry into rat brain regions. Biol Neonate 67:203–207

    Article  PubMed  CAS  Google Scholar 

  31. Hansen TWR (2000) Pioneers in the scientific study of neonatal jaundice and kernicterus. Pediatrics 106:e15

    Article  PubMed  CAS  Google Scholar 

  32. Hansen TWR, Maynard EC, Cashore WJ, Oh W (1993) Endotoxemia and brain bilirubin in the rat. Biol Neonate 63:171–176

    PubMed  CAS  Google Scholar 

  33. Haydar TF, Kuan CY, Flavell RA, Rakic P (1999) The role of cell death in regulating the size and shape of the mammalian forebrain. Cereb Cortex 9:621–626

    Article  PubMed  CAS  Google Scholar 

  34. Hertz L, Dringen R, Schousboe A, Robinson SR (1999) Astrocytes: glutamate producers for neurons. J Neurosci Res 57:417–428

    Article  PubMed  CAS  Google Scholar 

  35. Hong HN, Yoon SY, Suh J, Lee JH, Kim D (2002) Differential activation of caspase-3 at two maturational stages during okadaic acid-induced rat neuronal death. Neurosci Lett 334:63–67

    Article  PubMed  CAS  Google Scholar 

  36. Hutchins JB, Barger SW (1998) Why neurons die: cell death in the nervous system. Anat Rec 253:79–90

    Article  PubMed  CAS  Google Scholar 

  37. Kaplan M, Hammerman C (2004) Understanding and preventing severe neonatal hyperbilirubinemia: is bilirubin neurotoxity really a concern in the developed world? Clin Perinatol 31:555–575

    Article  PubMed  Google Scholar 

  38. Kaplan M, Hammerman C (2005) Understanding severe neonatal hyperbilirubinemia and preventing kernicterus: adjuncts in the interpretation of neonatal serum bilirubin. Clin Chim Acta 356:9–21

    Article  PubMed  CAS  Google Scholar 

  39. Kawade N, Onishi S (1981) The prenatal and postnatal development of UDP-glucuronyltransferase activity towards bilirubin and the effect of premature birth on this activity in the human liver. Biochem J 196:257–260

    PubMed  CAS  Google Scholar 

  40. Lin S, Yan C, Wei X, Paul SM, Du Y (2003) p38 MAP kinase mediates bilirubin-induced neuronal death of cultured rat cerebellar granule neurons. Neurosci Lett 353:209–212

    Article  PubMed  CAS  Google Scholar 

  41. Lucey JF (1972) The unsolved problem of kernicterus in the susceptible low birth weight infant. Pediatrics 49:646–647

    PubMed  CAS  Google Scholar 

  42. Mattson MP, Culmsee C, Yu Z, Camandola S (2000) Roles of nuclear factor κB in neuronal survival and plasticity. J Neurochem 74:443–456

    Article  PubMed  CAS  Google Scholar 

  43. McCarthy KD, de Vellis J (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85:890–902

    Article  PubMed  CAS  Google Scholar 

  44. McDonagh AF, Assisi F (1972) The ready isomerization of bilirubin IX-α in aqueous solution. Biochem J 129:797–800

    PubMed  CAS  Google Scholar 

  45. Mehler MF, Kessler JA (1997) Hematolymphopoietic and inflammatory cytokines in neural development. Trends Neurosci 20:357–365

    Article  PubMed  CAS  Google Scholar 

  46. Miyaoka T, Seno H, Itoga M, Iijima M, Inagaki T, Horiguchi J (2000) Schizophrenia-associated idiopathic unconjugated hyperbilirubinemia (Gilbert’s syndrome). J Clin Psychiatry 61:868–871

    PubMed  CAS  Google Scholar 

  47. Muñoz-Fernandez MA, Armas-Portela R, Diaz-Nido J, Alonso JL, Fresno M, Avila J (1991) Differential effects of tumor necrosis factor on the growth and differentiation of neuroblastoma and glioma cells. Exp Cell Res 194:161–164

    Article  PubMed  Google Scholar 

  48. Muñoz-Fernandez MA, Cano E, O’Donnell CA, Doyle J, Liew FY, Fresno M (1994) Tumor necrosis factor-α (TNF-α), interferon-γ, and interleukin-6 but not TNF-β induce differentiation of neuroblastoma cells: the role of nitric oxide. J Neurochem 62:1330–1336

    Article  PubMed  Google Scholar 

  49. Nakamura Y, Ohmaki M, Murakami K, Yoneda Y (2003) Involvement of protein kinase C in glutamate release from cultured microglia. Brain Res 962:122–128

    Article  PubMed  CAS  Google Scholar 

  50. Notter MF, Kendig JW (1986) Differential sensitivity of neural cells to bilirubin toxicity. Exp Neurol 94:670–682

    Article  PubMed  CAS  Google Scholar 

  51. O’Neill LA, Kaltschmidt C (1997) NF-κB: a crucial transcription factor for glial and neuronal cell function. Trends Neurosci 20:252–258

    Article  PubMed  CAS  Google Scholar 

  52. Oh W, Tyson JE, Fanaroff AA, Vohr BR, Perritt R, Stoll BJ, Ehrenkranz RA, Carlo WA, Shankaran S, Poole K, Wright LL (2003) Association between peak serum bilirubin and neurodevelopmental outcomes in extremely low birth weight infants. Pediatrics 112:773–779

    Article  PubMed  Google Scholar 

  53. Oh HL, Seok JY, Kwon CH, Kang SK, Kim YK (2006) Role of MAPK in ceramide-induced cell death in primary cultured astrocytes from mouse embryonic brain. Neurotoxicology 27:31–38

    Article  PubMed  CAS  Google Scholar 

  54. Ostrow JD, Pascolo L, Brites D, Tiribelli C (2004) Molecular basis of bilirubin-induced neurotoxicity. Trends Mol Med 10:65–70

    Article  PubMed  CAS  Google Scholar 

  55. Perlman JM, Rogers BB, Burns D (1997) Kernicteric findings at autopsy in two sick near term infants. Pediatrics 99:612–615

    Article  PubMed  CAS  Google Scholar 

  56. Porter ML, Dennis BL (2002) Hyperbilirubinemia in the term newborn. Am Fam Physician 65:599–606

    PubMed  Google Scholar 

  57. Pulsinelli WA (1985) Selective neuronal vulnerability: morphological and molecular characteristics. Prog Brain Res 63:29–37

    PubMed  CAS  Google Scholar 

  58. Ringheim GE, Burgher KL, Heroux JA (1995) Interleukin-6 mRNA expression by cortical neurons in culture: evidence for neuronal sources of interleukin-6 production in the brain. J Neuroimmunol 63:113–123

    Article  PubMed  CAS  Google Scholar 

  59. Ritter DA, Kenny JD, Norton HJ, Rudolph AJ (1982) A prospective study of free bilirubin and other risk factors in the development of kernicterus in premature infants. Pediatrics 69:260–266

    PubMed  CAS  Google Scholar 

  60. Rodrigues CMP, Solá S, Brites D (2002) Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology 35:1186–1195

    Article  PubMed  CAS  Google Scholar 

  61. Romijn HJ, Hofman MA, Gramsbergen A (1991) At what age is the developing cerebral cortex of the rat comparable to that of the full-term newborn human baby? Early Hum Dev 26:61–67

    Article  PubMed  CAS  Google Scholar 

  62. Roth KA, D’Sa C (2001) Apoptosis and brain development. Ment Retard Dev Disabil Res Rev 7:261–266

    Article  PubMed  CAS  Google Scholar 

  63. Rubaltelli FF, Griffith PF (1992) Management of neonatal hyperbilirubinaemia and prevention of kernicterus. Drugs 43:864–872

    Article  PubMed  CAS  Google Scholar 

  64. Sanz O, Acarin L, Gonzalez B, Castellano B (2002) NF-κB and IκBα expression following traumatic brain injury to the immature rat brain. J Neurosci Res 67:772–780

    Article  PubMed  CAS  Google Scholar 

  65. Sawada M, Suzumura A, Marunouchi T (1995) Cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells. Int J Dev Neurosci 13:253–264

    Article  PubMed  CAS  Google Scholar 

  66. Schobitz B, Voorhuis DA, De Kloet ER (1992) Localization of interleukin 6 mRNA and interleukin 6 receptor mRNA in rat brain. Neurosci Lett 136:189–192

    Article  PubMed  CAS  Google Scholar 

  67. Selmaj KW, Farooq M, Norton WT, Raine CS, Brosnan CF (1990) Proliferation of astrocytes in vitro in response to cytokines. A primary role for tumor necrosis factor. J Immunol 144:129–135

    CAS  Google Scholar 

  68. Shapiro SM (2005) Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J Perinatol 25:54–59

    Article  PubMed  CAS  Google Scholar 

  69. Silva R, Mata LR, Gulbenkian S, Brito MA, Tiribelli C, Brites D (1999) Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and pH. Biochem Biophys Res Commun 265:67–72

    Article  PubMed  CAS  Google Scholar 

  70. Silva RFM, Rodrigues CMP, Brites D (2002) Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin. Pediatr Res 51:535–541

    Article  PubMed  CAS  Google Scholar 

  71. Silva RFM, Falcão AS, Fernandes A, Gordo AC, Brito MA, Brites D (2006) Cell cultures as models for assessment of neurotoxicity. Toxicol Lett 163:1–9

    Article  PubMed  CAS  Google Scholar 

  72. Soorani-Lunsing I, Woltil HA, Hadders-Algra M (2001) Are moderate degrees of hyperbilirubinemia in healthy term neonates really safe for the brain? Pediatr Res 50:701–705

    Article  PubMed  CAS  Google Scholar 

  73. Stevenson DK, Dennery PA, Hintz SR (2001) Understanding newborn jaundice. J Perinatol 21(Suppl. 1):S21–S24

    Article  PubMed  Google Scholar 

  74. Tak PP, Firestein GS (2001) NF-κB: a key role in inflammatory diseases. J Clin Invest 107:7–11

    Article  PubMed  CAS  Google Scholar 

  75. Turkel SB, Miller CA, Guttenberg ME, Moynes DR, Godgman JE (1982) A clinical pathologic reappraisal of kernicterus. Pediatrics 69:267–272

    PubMed  CAS  Google Scholar 

  76. Watchko JF, Maisels MJ (2003) Jaundice in low birthweight infants: pathobiology and outcome. Arch Dis Child Fetal Neonatal Ed 88:F455–F458

    Article  PubMed  CAS  Google Scholar 

  77. Watchko JF, Daood MJ, Biniwale M (2002) Understanding neonatal hyperbilirubinaemia in the era of genomics. Semin Neonatol 7:143–152

    Article  PubMed  Google Scholar 

  78. Xu L, Chock VY, Yang EY, Giffard RG (2004) Susceptibility to apoptosis varies with time in culture for murine neurons and astrocytes: changes in gene expression and activity. Neurol Res 26:632–643

    Article  PubMed  CAS  Google Scholar 

  79. Yakovlev AG, Ota K, Wang G, Movsesyan V, Bao WL, Yoshihara K, Faden AI (2001) Differential expression of apoptotic protease-activating factor-1 and caspase-3 genes and susceptibility to apoptosis during brain development and after traumatic brain injury. J Neurosci 21:7439–7446

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants POCI/39906/FCB/2001 and POCI/SAU-MMO/55955/2004, from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal, and FEDER (to D.B.), and Ph.D. Fellowships SFRH/BD/8436/2002 and SFRH/BD/9204/2002 from FCT (to A.S.F. and A.F.)

Author information

Authors and Affiliations

  1. Centro de Patogénese Molecular-UBMBE, Faculdade de Farmácia, University of Lisbon, Av. Forças Armadas, 1600-083, Lisbon, Portugal

    Ana S. Falcão, Adelaide Fernandes, Maria A. Brito, Rui F. M. Silva & Dora Brites

Authors
  1. Ana S. Falcão
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adelaide Fernandes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria A. Brito
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rui F. M. Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dora Brites
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dora Brites.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Falcão, A.S., Fernandes, A., Brito, M.A. et al. Bilirubin-induced immunostimulant effects and toxicity vary with neural cell type and maturation state. Acta Neuropathol 112, 95–105 (2006). https://doi.org/10.1007/s00401-006-0078-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00401-006-0078-4

Keywords

Search

Navigation