, Volume 33, Issue 4, pp 497–507 | Cite as

Melatonin is able to delay endoplasmic reticulum stress-induced apoptosis in leukocytes from elderly humans

  • Javier Espino
  • Ignacio Bejarano
  • Sergio D. Paredes
  • Carmen Barriga
  • Russel J. Reiter
  • José A. Pariente
  • Ana B. RodríguezEmail author


The mechanisms regulating neutrophil apoptosis are basically unaffected by the aging process. However, a significant impairment of cell survival occurs in elderly individuals following neutrophil challenge with pro-inflammatory stimuli, such as granulocyte-macrophage colony-stimulating factor (GM-CSF). The goal of the present study was to prove the effects of melatonin supplementation on apoptosis induced by calcium signaling in human leukocytes from elderly volunteers. Treatments with the specific inhibitor of cytosolic calcium re-uptake, thapsigargin, and/or the calcium mobilizing agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced mitochondrial membrane depolarization, caspase activation, phosphatidylserine (PS) externalization, and DNA fragmentation in leukocytes from both young and elderly volunteers, although such effects were much more evident in aged leukocytes. Importantly, melatonin treatment substantially preserved mitochondrial membrane potential, reversed caspase activation, reduced PS exposure and forestalled DNA fragmentation in leukocytes from both age groups. In conclusion, melatonin is able to delay endoplasmic reticulum stress-induced apoptosis in aged leukocytes and may counteract, at the cellular level, age-related degenerative phenomena linked to oxidative stress.


Melatonin Aging Leukocytes Apoptosis 



This work was supported by Junta de Extremadura (PRI07-A024) and MEC-Fondo Social Europeo de Desarrollo Regional grants (BFU2007-60091 and BFU2010-15049). I. Bejarano and S.D. Paredes are beneficiaries of grants from Consejería de Economía, Comercio e Innovación – Fondo Social Europeo (Junta de Extremadura, PRE06070 and POS07012, respectively). J. Espino is beneficiary of grant from MEC (AP2009-0753).


  1. Antonaci S, Jirillo E, Ventura MT, Garofalo AR, Bonomo L (1984) Nonspecific immunity in aging: deficiency of monocyte and polymorphonuclear cell-mediated functions. Mech Ageing Dev 24:367–375PubMedCrossRefGoogle Scholar
  2. Bejarano I, Lozano GM, Ortiz A, García JF, Paredes SD, Rodríguez AB, Pariente JA (2008) Caspase 3 activation in human spermatozoa in response to hydrogen peroxide and progesterone. Fertil Steril 90:1340–1347PubMedCrossRefGoogle Scholar
  3. Bejarano I, Redondo PC, Espino J, Rosado JA, Paredes SD, Barriga C, Reiter RJ, Pariente JA, Rodríguez AB (2009) Melatonin induces mitochondrial-mediated apoptosis in human myeloid HL-60 cells. J Pineal Res 46:392–400PubMedCrossRefGoogle Scholar
  4. Benot S, Goberna R, Reiter RJ, Garcia-Maurino S, Osuna C, Guerrero JM (1999) Physiological levels of melatonin contribute to the antioxidant capacity of human serum. J Pineal Res 27:59–64PubMedCrossRefGoogle Scholar
  5. Butcher SK, Chahal H, Nayak L, Sinclair A, Henriquez NV, Sapey E, O’Mahony D, Lord JM (2001) Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans. J Leukoc Biol 70:881–886PubMedGoogle Scholar
  6. Carretero M, Escames G, López LC, Venegas C, Dayoub JC, García L, Acuña-Castroviejo D (2009) Long-term melatonin administration protects brain mitochondria from aging. J Pineal Res 47:192–200PubMedCrossRefGoogle Scholar
  7. Espino J, Bejarano I, Paredes SD, González D, Barriga C, Reiter RJ, Pariente JA, Rodriguez AB (2010a) Melatonin counteracts alterations in oxidative metabolism and cell viability induced by intracellular calcium overload in human leukocytes: changes with age. Basic Clin Pharmacol Toxicol 107:590–597PubMedCrossRefGoogle Scholar
  8. Espino J, Bejarano I, Redondo PC, Rosado JA, Barriga C, Reiter RJ, Pariente JA, Rodriguez AB (2010b) Melatonin reduces caspase activation induced by calcium signaling in human leucocytes: evidence for the involvement of mitochondria and Bax activation. J Membr Biol 233:105–118PubMedCrossRefGoogle Scholar
  9. García JJ, Reiter RJ, Pié J, Ortiz GG, Cabrera J, Sainz RM, Acuña-Castroviejo D (1999) Role of pinoline and melatonin in stabilizing hepatic microsomal membranes against oxidative stress. J Bioenerg Biomembr 6:609–616CrossRefGoogle Scholar
  10. Gavazzi G, Krauze KH (2002) Ageing and infection. Lancet Infect Dis 2:659–666PubMedCrossRefGoogle Scholar
  11. Genestier AL, Michallet MC, Prévost G, Bellot G, Chalabreysse L, Peurol S, Thivolet F, Etienne J, Lina G, Vallette FM, Vandenesch F, Genestier L (2005) Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils. J Clin Invest 115:3117–3127PubMedCrossRefGoogle Scholar
  12. Gutierrez-Cuesta J, Tajes M, Jiménez A, Coto-Montes A, Camins A, Pallàs M (2008) Evaluation of potential pro-survival pathways regulated by melatonin in a murine senescence model. J Pineal Res 45:497–505PubMedCrossRefGoogle Scholar
  13. Harman D (1992) Free radical theory of aging. Mutat Res 275:257–266PubMedGoogle Scholar
  14. Heatwole VM (1999) TUNEL assay for apoptotic cells. Methods Mol Biol 115:141–148PubMedGoogle Scholar
  15. Juknat AA, Mendez Mdel V, Quaglino A, Fameli CI, Mena M, Kotler ML (2005) Melatonin prevents hydrogen peroxide-induced bax expression in cultured rat astrocytes. J Pineal Res 38:84–92PubMedCrossRefGoogle Scholar
  16. Larbi A, Douziech N, Fortin C, Linteau A, Dupuis G, Fulop T Jr (2005) The role of the MAPK pathway alterations in GM-CSF modulated human neutrophil apoptosis with aging. Immun Ageing 2:6. doi: 10.1186/1742-4933-2-6 PubMedCrossRefGoogle Scholar
  17. León J, Acuña-Castroviejo D, Sainz RM, Mayo JC, Tan DX, Reiter RJ (2004) Melatonin and mitochondrial function. Life Sci 75:765–790PubMedCrossRefGoogle Scholar
  18. León J, Acuña-Castroviejo D, Escames G, Tan DX, Reiter RJ (2005) Melatonin mitigates mitochondrial malfunction. J Pineal Res 38:1–9PubMedCrossRefGoogle Scholar
  19. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489PubMedCrossRefGoogle Scholar
  20. Luchetti F, Betti M, Canonico B, Arcangeletti M, Ferri P, Galli F, Papa S (2009) ERK MAPK activation mediates the antiapoptotic signalling of melatonin in UVB-stressed U937 cells. Free Radic Biol Med 46:339–351PubMedCrossRefGoogle Scholar
  21. Martín M, Macías M, Escames G, Reiter RJ, Agapito MT, Ortiz GG, Acuña-Castroviejo D (2000) Melatonin-induced increased activity of the respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium red in vivo. J Pineal Res 28:242–248PubMedCrossRefGoogle Scholar
  22. Mayo JC, Sainz RM, Uria H, Antolín I, Estéban MM, Rodríguez C (1998) Inhibition of cell proliferation: a mechanism likely to mediate the prevention of neuronal cell death by melatonin. J Pineal Res 25:12–18PubMedCrossRefGoogle Scholar
  23. Mazzoccoli G, Vendemiale G, La Viola M, De Cata A, Carughi S, Greco A, Balzanelli M, Tarquini R (2010) Circadian variations of cortisol, melatonin and lymphocyte subpopulations in geriatric age. Int J Immunopathol Pharmacol 23:289–296PubMedGoogle Scholar
  24. Miyoshi N, Oubrahim H, Chock PB, Stadtman ER (2006) Age-dependent cell death and the role of ATP in hydrogen peroxide-induced apoptosis and necrosis. Proc Natl Acad Sci USA 103:1727–1731PubMedCrossRefGoogle Scholar
  25. Orrenius S, Zhivotovsky B, Nicotera P (2003) Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4:552–565PubMedCrossRefGoogle Scholar
  26. Otton R, da Silva DO, Campoio TR, Silveira LR, de Souza MO, Hatanaka E, Curi R (2007) Non-esterified fatty acids and human lymphocyte death: a mechanism that involves calcium release and oxidative stress. J Endocrinol 195:133–143PubMedCrossRefGoogle Scholar
  27. Paradies G, Petrosillo G, Paradies V, Reiter RJ, Ruggiero FM (2010) Melatonin, cardiolipin and mitochondrial bioenergetics in health and disease. J Pineal Res 48:297–310PubMedCrossRefGoogle Scholar
  28. Paredes SD, Bejarano I, Terrón MP, Barriga C, Reiter RJ, Rodríguez AB (2009) Melatonin and tryptophan counteract lipid peroxidation and modulate superoxide dismutase activity in ringdove heterophils in vivo. Effect of antigen-induced activation and age. Age 31:179–188PubMedCrossRefGoogle Scholar
  29. Poeggeler B (2005) Melatonin, aging, and age-related diseases: perspectives for prevention, intervention, and therapy. Endocr 27:201–212CrossRefGoogle Scholar
  30. Pollack M, Leeuwenburgh C (2001) Apoptosis and aging: role of mitochondria. J Gerontol A-Biol 11A:B475–B482CrossRefGoogle Scholar
  31. Reiter RJ (1992) The ageing pineal gland and its physiological consequences. Bioessays 14:169–175PubMedCrossRefGoogle Scholar
  32. Reiter RJ, Tan DX (2003) Melatonin: a novel protective agent against oxidative injury of the ischemic/reperfused heart. Cardiovasc Res 58:10–19PubMedCrossRefGoogle Scholar
  33. Reiter RJ, Tan DX, Manchester LC, Qi W (2001) Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of the evidence. Cell Biochem Biophys 34:237–256PubMedCrossRefGoogle Scholar
  34. Reiter RJ, Tan DX, Manchester LC, El-Sawi MR (2002) Melatonin reduces oxidant damage and promotes mitochondrial respiration: implications for aging. Ann NY Acad Sci 959:238–250PubMedCrossRefGoogle Scholar
  35. Reiter RJ, Paredes SD, Korkmaz A, Manchester LC, Tan DX (2008) Melatonin in relation to the ‘strong’ and ‘weak’ versions of the free radical theory of aging. Adv Med Sci 53:119–129PubMedCrossRefGoogle Scholar
  36. Reiter RJ, Paredes SD, Manchester LC, Tan DX (2009) Reducing oxidative/nitrosative stress: a newly-discovered genre for melatonin. Crit Rev Biochem Mol Biol 44:175–200PubMedCrossRefGoogle Scholar
  37. Semsei I (2000) On the nature of aging. Mech Ageing Dev 117:93–108PubMedCrossRefGoogle Scholar
  38. Sies H (1985) Oxidative stress: introductory remarks. In: Sies H (ed) Oxidative stress. Academic, London, pp 1–8Google Scholar
  39. Tajes Orduña M, Pelegrí Gabalda C, Vilaplana Hortensi J, Pallàs Lliberia M, Camins Espuny A (2009) An evaluation of the neuroprotective effects of melatonin in an in vitro experimental model of age-induced neuronal apoptosis. J Pineal Res 46:262–267PubMedCrossRefGoogle Scholar
  40. Tan DX, Chen LD, Poeggeler B, Manchester LC, Reiter RJ (1993) Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr J 1:57–60Google Scholar
  41. Terrón MP, Marchena JM, Shadi F, Harvey S, Lea RW, Rodríguez AB (2001) Melatonin: an antioxidant at physiological concentrations. J Pineal Res 31:95–96PubMedCrossRefGoogle Scholar
  42. Tortorella C, Piazzolla G, Spaccavento F, Pece S, Jirillo E, Antonaci S (1998) Spontaneous and Fas-induced apoptotic cell death in aged neutrophils. J Clin Immunol 18:321–329PubMedCrossRefGoogle Scholar
  43. Tortorella C, Simone O, Piazzolla G, Stella I, Cappiello V, Antonaci S (2006) Role of phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways in granulocyte macrophage-colony-stimulating factor failure to delay Fas-induced neutrophil apoptosis in elderly humans. J Gerontol A-Biol 61:1111–1118Google Scholar
  44. Wenisch C, Patruta S, Daxbock F, Krause R, Horl W (2000) Effect of age on human neutrophil function. J Leukoc Biol 67:40–45PubMedGoogle Scholar
  45. Whyte MKB, Meagher LC, MacDermot J, Haslett C (1993) Impairment of function in aging neutrophils is associated with apoptosis. J Immunol 150:5124–5134PubMedGoogle Scholar
  46. Zhang Y, Herman B (2002) Ageing and apoptosis. Mech Ageing Dev 123:245–260PubMedCrossRefGoogle Scholar
  47. Zhang Y, Chong E, Herman B (2002) Age-associated increases in the activity of multiple caspases in Fisher 344 rat organs. Exp Gerontol 37:777–789PubMedCrossRefGoogle Scholar
  48. Zhang JH, Zhang Y, Herman B (2003) Caspases, apoptosis and aging. Ageing Res Rev 2:357–366PubMedCrossRefGoogle Scholar

Copyright information

© American Aging Association 2010

Authors and Affiliations

  • Javier Espino
    • 1
  • Ignacio Bejarano
    • 1
  • Sergio D. Paredes
    • 1
  • Carmen Barriga
    • 1
  • Russel J. Reiter
    • 2
  • José A. Pariente
    • 1
  • Ana B. Rodríguez
    • 1
    Email author
  1. 1.Department of Physiology, Neuroimmunophysiology and Chrononutrition Research Group, Faculty of ScienceUniversity of ExtremaduraBadajozSpain
  2. 2.Department of Cellular & Structural BiologyUniversity of Texas Health Science CenterSan AntonioUSA

Personalised recommendations