- Email: [email protected]
Effects of a reduced oxygen tension culture system on human T cell clones as a function of in vitro age
Experimental Gerontology 39 (2004) 525–530 www.elsevier.com/locate/expgero
Effects of a reduced oxygen tension culture system on human T cell clones as a function of in vitro age Orla Duggana, Paul Hylanda, Kathryn Annetta, Robin Freeburna, Christopher Barnettb, Graham Pawelecc, Yvonne Barnetta,* a
Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK b Research Support Office, Trent Building, University Park, University of Nottingham, Nottingham NG7 2RD, UK c University of Tu¨bingen, Tu¨bingen Ageing and Tumour Immunology Group (TATI), ZMF, Waldho¨rnlestr. 22, D-72072 Tu¨bingen, Germany Received 14 July 2003; received in revised form 26 November 2003; accepted 1 December 2003
Abstract Oxidative DNA damage has been suggested to contribute to the decline in T cell clone (TCC) function with increased age in vitro. To test this hypothesis the effect of a reduced oxygen tension culture system (6% O2) on TCCs was examined. Specifically, the effects of the altered culture conditions on DNA damage levels, in vitro lifespan and proliferative capacity were assessed in five independently derived human CD4 þ TCCs. DNA damage levels over the entire lifespan were significantly lowered by reducing oxygen tension. Lifespan (total population doublings (PDs) achieved) and proliferative capacity (PDs/week) were reduced for all clones under reduced oxygen tension when compared to standard culture conditions. This observed tendency warrants further investigation using a greater number of clones from donors of all age groups before definitive conclusions regarding the effect of low oxygen tension on the lifespan and proliferative capacity of TCC can be made. However, these results may suggest that the reduced oxygen tension culture system has interfered with some aspect of T cell biology not yet examined within the remit of this study. q 2004 Elsevier Inc. All rights reserved. Keywords: Reduced oxygen tension; T cell clones; Oxidative DNA damage; Lifespan; Proliferative capacity
1. Introduction T cells in vivo are exposed to reactive oxygen species (ROS) from both intrinsic (e.g. mitochondria, oxidases, peroxisomes) and extrinsic sources (e.g. radiation, pollution, xenobiotics) (Pacifici and Davies, 1991; Ames et al., 1993). At sites of inflammation, cells of the immune system are exposed to high levels of ROS, which are produced as part of a normal immune response. ROS produced in excess may cause damage to T cell biomolecules and so result in altered T cell function or loss. Previous investigations within our laboratory found that in ex vivo T cells an increase in levels of genetic damage as a function of age (King et al., 1994, 1997; Barnett and King, 1995) and in human T cell clones (TCCs) cultured under 20% O2 tension levels of oxidative DNA damage increased in TCCs with * Corresponding author. Tel.: þ44-28-7032-4163; fax: þ 44-28-70324965. E-mail address: [email protected] (Y. Barnett). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.12.010
increasing time in culture (Hyland et al., 2000, 2001). Apart from the potentially detrimental effects of cellular biomolecules, ROS are also important as intracellular signalling molecules within T cells (Kamata and Hirata, 1999; Schreck et al., 1992). In this study we have examined the effect of reduced oxygen tension culture conditions on human CD4 þ TCCs. Proliferative capacity, lifespan and levels of DNA damage were examined in the TCCs grown under reduced oxygen tension throughout their lifespan.
2. Materials and methods 2.1. Culture of peripheral blood derived human CD4 þ T cell clones Five independently derived human TCCs were examined. The 400 series clones were obtained from
526
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
a 26-year-old overtly healthy laboratory worker, the 385 series clones were derived from an overtly healthy 45-yearold adult donor, and the 399 series clones were derived from a healthy 80-year-old donor conforming to the SENIEUR protocol for healthily aged individuals. Clones were grown in a serum-free medium (2 ml per well), X-Vivo 15 (BioWhittaker), at concentrations of 4 £ 105 cells per well, along with 2 £ 105 gamma-irradiated (80 Gy) RJK853 cells per well (EBV-transformed B-lymphoblastoid cell line with complete hprt deletion, a gift from Dr Jane Cole, MRC Cell Mutation Unit, University of Sussex, UK), as feeder cells. Clones maintained in standard culture conditions were incubated at 37 8C in a 5% CO2, 21% O2, 74% N2 atmosphere in a 7-day cycle. On days 1 and 4 of the cycle the clones were supplemented with 400 U/ml recombinant human IL-2 (Aldesleukin, Chiron, UK). On day 7 of the cycle, the cells were harvested and a viable cell count was performed using an improved Neubauer Counting Chamber, and a new culture cycle was set up with fresh medium and RJK853 feeder cells. At the end of each 7-day growth cycle, samples were removed and cryopreserved in a medium made up of 10% DMSO, 20% foetal bovine serum and 70% X-Vivo 15 and stored in liquid nitrogen. 2.2. Reduced oxygen tension culture conditions Reduced oxygen tension culture conditions were achieved by incubating the clones at 37 8C in a 3% O2, 5% CO2, 92% N2 atmosphere for 10 weeks and then at 37 8C in a 6% O2, 5% CO2, 89% N2 atmosphere until the end of the lifespans of the clones. The TCCs were shifted to a higher oxygen tension after 10 weeks because there was no measurable proliferation at 3% O2 although the clones remained viable. 2.3. Determination of T cell clone lifespan and proliferative capacity TCC proliferative capacity was calculated by determining the number of population doublings (PDs) achieved by each clone, per week, using the formula: PD ¼ log2(cell count at end of cycle) 2 log2(number of cells at start of cycle) Lifespan for each TCC was determined by the total cumulative PDs achieved. 2.4. Determination of oxidative DNA damage levels in T cell clones Levels of oxidative DNA damage in TCCs grown were determined at three time points during their in vitro lifespans. Details of the modified alkaline comet assay used have been described previously (Collins et al., 1993; Hyland et al., 2000, 2001). The modified comet assay uses formamidopyrimidine glycosylase, which recognises
Table 1 Total population doublings achieved by TCCs maintained under different culture conditions Clone
Standard oxygen tension
Reduced oxygen tension
385-2 385-7 399-35 399-37 400-23 Mean lifespan (PD)
69.9 73.5 72.1 78.1 80.7 74.9 ^ 4.4
61.6 71.5 45.0 51.9 51.7 56.3 ^ 10.3*
*Significantly lower, p , 0:05:
oxidatively modified purines (Boiteux et al., 1992) and endonuclease III (Endo III) which recognises oxidatively modified pyrimidines (Asahara et al., 1989). These enzymes nick DNA at the sites of oxidatively damaged nucleotides, creating single strand-breaks, which can be detected with the assay. Comet analysis was performed using Komet 3.0 analysis software (Kinetic Imaging, UK), counting 50 cells per slide. DNA damage results were expressed as the percentage of DNA in the comet tail. 2.5. Statistical analysis Lifespan and proliferative capacity results were tested for significance using paired 2-sample 1-tailed Student’s t-tests assuming equal variance.
3. Results 3.1. T cell clone lifespan and proliferative capacity Table 1 shows the cumulative PDs achieved by each clone and the average lifespan achieved overall in each culture condition. The results in this table show that clones grown under the reduced oxygen conditions had shorter lifespans than their respective controls ðp , 0:05Þ: Reduced oxygen conditions had the greatest effect on lifespans in TCC from the young and old donors, whereas the TCC from the middle-aged donor were minimally effected. Table 2 details the mean proliferative Table 2 Mean proliferative capacity per week (PDs/week) for each T cell clone maintained under different culture conditions Clone
Standard oxygen tension
Reduced oxygen tension
385-2 385-7 399-35 399-37 400-23 Mean PD/week
1.9 ^ 0.6 2.1 ^ 0.7 2.1 ^ 0.9 2.2 ^ 0.8 2.1 ^ 1.0 2.1 ^ 0.1
1.1 ^ 0.6 1.2 ^ 0.6 0.8 ^ 0.5 0.9 ^ 0.6 0.9 ^ 0.6 1.0 ^ 0.2*
*Significantly lower, p , 0:05:
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
capacity per week for each TCC under each of the culture conditions. The results presented in Table 2 show that clones grown under reduced oxygen tension also had reduced replicative capacity compared to controls ðp , 0:05Þ:
527
3.2. Oxidative DNA damage levels in T cell clones Fig. 1 illustrates the levels of DNA damage as a function of in vitro age in TCCs grown under standard or reduced oxygen conditions. From this figure it can be seen
Fig. 1. Oxidative DNA damage levels in T cell clones maintained under standard and reduced oxygen tension conditions. (a) DNA strand breaks and alkali labile sites, (b) oxidatively damaged pyrimidine bases plus DNA strand breaks and alkali labile sites and (c) oxidatively damaged purine bases plus DNA strand breaks and alkali labile sites.
528
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
that throughout the lifespan of the clones, DNA damage levels in those cultured under reduced oxygen tension were maintained at lower levels when compared to the same clones grown under standard conditions. Fig. 1(a) shows the DNA strand breaks and alkali labile sites present throughout the lifespan of the TCCs under both culture conditions. Fig. 1(b) shows the DNA strand breaks and alkali labile sites plus oxidative DNA damage to pyrimidine bases and Fig. 1(c) shows the DNA strand breaks and alkali labile sites plus oxidative DNA damage to purine bases under both reduced and standard oxygen tensions. Fig. 1(b) and (c) show that the levels of oxidative DNA damage plus DNA strand breaks and alkali labile sites remain low throughout the lifespan of the TCCs cultured under reduced oxygen tension. In comparison, the control clones cultured under standard oxygen tension showed an increase in all aspects of oxidative DNA damage levels with in vitro age. Donor age appeared to exert no effect on the observed levels of DNA damage. This is in accordance with previously reported studies (Hyland et al., 2000, 2001).
4. Discussion The free radical theory of ageing (Harman, 1956) suggested an important role for ROS in the aetiology of ageing and age-related pathologies. Since this theory was first reported much evidence has been found to support it (Beckman and Ames, 1998). A number of investigations have indicated that there may be a relationship between oxygen tension (and thus ROS levels), cellular senescence and lifespan. The results of previous studies have suggested a beneficial effect of reduced oxygen tension on human diploid fibroblast (HDF) cultures. Packer and Feuher (1977) found that growing two types of HDFs (WI-37 and IMR-90) under 10% oxygen extended their lifespans (measured in total PDs) by 25%. More recently, Chen et al. (1995) have demonstrated that long-term culture of HDFs under a more physiological oxygen concentration of 3% O2 could significantly extend the in vitro lifespan of the fibroblasts. The HDFs grown under reduced oxygen tension attained approximately 50% more PDs during their in vitro lifespan. It was also noted that HDFs cultured under reduced oxygen tension had increased rates of proliferation compared to cells cultured under standard 20% O2. Chen et al. also reported that the reduced oxygen tension helped to maintain a juvenile HDF phenotype. The converse effect has also been demonstrated by von Zglinicki et al. (1995), where the replicative lifespan of HDFs were shortened under increased oxygen tension of 40% O2. The beneficial effect of a low oxygen tension environment is likely to be due to reduced free radical levels under such conditions, leading to lowered levels of radical induced biomolecule damage. In view of the fact that TCCs grown under standard (20% O2) culture conditions accumulate oxidative DNA
damage with age (Hyland et al., 2000, 2001), the potential beneficial effects of lowered oxygen tensions on TCCs were investigated. The results of the present study demonstrated that levels of oxidative DNA damage were maintained at a relatively low level throughout the lifespan of the clones grown under reduced oxygen tension. Standard TCC cultures exhibited an age-related increase in the levels of oxidative DNA damage. However, the TCC in this study had shorter lifespans and reduced replicative capacities under reduced oxygen tension when compared to the clones grown under standard culture conditions. This effect appeared to be greater in TCC from the young and old donor, and less in the clone from the middle-aged donor. This may not be significant as it has been established that TCC longevity is independent of donor age (McCarron et al., 1987; Pawelec et al., 2002). This is in agreement with recent work done on human fibroblasts, where the long-established view that fibroblast longevity in culture was dependent on donor age was shown not to be the case when donor health status and biopsy conditions were rigorously controlled (Cristofalo et al., 1998). However, this observed tendency towards impaired proliferation warrants further investigation using a greater number of clones from donors of all age groups before any definitive relationship between low oxygen tension culture systems, donor age, lifespan and proliferative capacity of TCC can be proposed. These observations on lifespan in TCC conflict with the previous HDF studies. There may be basic differences in the biology of ageing in fibroblasts and T cells. For example, recent studies have shown that telomere lengths in fibroblasts ex vivo are not decreased in the elderly, up to centenarian age, although the same fibroblasts did show telomere length decreases upon in vitro culture (Mondello et al., 1999). In contrast, telomere length in blood cells ex vivo was shown to be related to donor age (Slagboom et al., 1994), even in the same individuals mentioned above, whose fibroblasts failed to show a decrease, although there is a great deal of inter-individual variation (Mondello et al., 1999). In addition TCC are known to be more susceptible to variations in culture conditions than fibroblast cultures (Pawelec et al., 2002). Results from previous investigations on senescence and longevity were derived from polyclonal fibroblast populations, which behave differently from the monoclonal TCC populations. Not only do these two different cell types behave differently under similar conditions, but the fact that HDFs are polyclonal means that the studies are examining the behaviour of the longest-lived clones within that population. However, when using the TCC culture system the behaviour of each single cell type is investigated. It is possible that polyclonal T cell populations studied under these same conditions may behave in a similar manner to the HDFs, and this is currently under investigation within our laboratory.
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
It seems probable that some aspect of T cell biology not yet examined within the remit of this study may have been affected by the reduced oxygen tension culture conditions. This interference could explain the reasons for the reduced proliferation of the TCCs grown under reduced oxygen tensions, and may have contributed the reduced lifespans of these TCCs. Although ROS are typically thought of as harmful molecules, which cause cellular damage, they are also known to be important intracellular signalling molecules within T cells (Kamata and Hirata, 1999; Schreck et al., 1992). Examples are the requirement of ROS for cell proliferation induced by lectin, and the involvement of ROS in T cell signalling events such as protein tyrosine phosphorylation and the activation of JNK1 (Pani et al., 2000). Therefore one possible reason for the observed reduced proliferation in TCC may be that the 3/6% oxygen culture conditions may have resulted in a reduced intracellular level of oxygen free radicals and so may have altered the overall redox balance of the TCCs. The lack of an age-related increase in oxidative DNA damage levels in clones grown under reduced oxygen tension, may provide evidence of lower levels of oxygen-derived free radicals in these clones. In turn, lowered levels of oxygen-derived free radicals may result in an interference with signalling pathways, e.g. the redox-sensitive activation of transcription factors such as NFkB or AP-1, involved in T cell activation and proliferation (Tatla et al., 1998). This interference may have contributed to the reduced proliferation and reduced lifespans of the TCCs cultured under reduced oxygen tensions. The results of this study clearly warrant further investigation. We are currently investigating the effects of various concentrations of oxygen tension on T cell biology. Both monoclonal and polyclonal T cell populations are being included in these analyses. In addition, it is necessary to investigate other aspects of T cell biology such as signalling pathways effected by oxygen tension. The preliminary results of this study highlight that any interventions aimed at reducing the accumulation of oxidative damage with age in vivo in T cells (or other cell types) should be carefully optimised to ensure that normal cellular activities can still occur and that there are no other detrimental effects on other aspects of the target cell biology.
Acknowledgements The authors wish to acknowledge: University of Ulster for a Vice Chancellors Research Studentship to OD; The Department of Education and Learning for a Studentship to KA; The European Union for funding support under the aegis of Immunology and Ageing in Europe (ImAginE; QLK6-CT-1999-O2031) and T cell Immunity and Ageing (TCIA; QL1C6-CT-2002-02283).
529
References Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants and the degenerative diseases of aging. Proc. Natl Acad. Sci. USA 90, 7915–7922. Asahara, H., Wisort, P.M., Bank, J.F., Bakerian, R.H., Cunningham, R.P., 1989. Purification and characterisation of Escherichia coli endonuclease III from the cloned nth gene. Biochemistry 28, 4444–4449. Barnett, Y.A., King, C.M., 1995. An investigation of antioxidant status, DNA repair capacity and mutation as a function of age in humans. Mutat. Res. 338, 115 –128. Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 70 (2), 547– 581. Boiteux, S., Gajewski, E., Laval, J., Dizdaroglu, M., 1992. Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine–DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization. Biochemistry 31, 106– 110. Chen, Q., Fischer, A., Reagan, J.D., Yan, L.-J., Ames, B.N., 1995. Oxidative DNA damage and senescence of human diploid fibroblast cells. Proc. Natl Acad. Sci. USA 92, 4337–4341. Collins, A.R., Duthie, S.J., Dobson, V.L., 1993. Direct enzymatic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14, 1733–1735. Cristofalo, V.J., Allen, R.G., Pignolo, R.J., Martin, B.G., Beck, J.C., 1998. Relationship between donor age and the replicative lifespan of human cells in culture: a re-evaluation. Proc. Natl Acad. Sci. USA. 95, 10614–10619. Harman, D., 1956. Ageing: a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300. Hyland, P., Duggan, O., Hipkiss, A., Barnett, C., Barnett, Y., 2000. The effects of carnosine on oxidative DNA damage levels and in vitro lifespan in human peripheral blood derived CD4 þ T cell clones. Mech. Ageing Dev. 121, 203–215. Hyland, P., Barnett, C., Pawelec, G., Barnett, Y., 2001. Age-related accumulation of oxidative DNA damage and alterations in levels of p16INK4a/CDKN2a, p21WAF1/CIP1/SDI1 and p27KIP1 in human CD4 þ T cell clones in vitro. Mech. Ageing Dev. 122 (11), 1151–1167. Kamata, H., Hirata, H., 1999. Redox regulation of cellular signalling. Cell. Signal. 11 (1), 1–14. King, C.M., Gillespie, E.S., McKenna, P.G., Barnett, Y.A., 1994. An investigation of mutation as a function of age in humans. Mutat. Res.DNAging Genet. Instabil. Aging 316 (2), 79 –90. King, C.M., Bristow-Craig, H.E., Gillespie, E.S., Barnett, Y.A., 1997. In vivo antioxidant status, DNA damage, mutation and DNA repair capacity in cultured lymphocytes from healthy 75–80 year old humans. Mutat. Res. 377, 137 –147. McCarron, M., Osborne, Y., Story, C., Dempsey, J.L., Turner, R., Morley, A., 1987. Effect of age on lymphocyte proliferation. Mech. Ageing Dev. 41, 211–218. Mondello, C., Petropoulou, C., Monti, D., Gonos, E.S., Franceschi, C., Nuzzo, F., 1999. Telomere length in fibroblasts and blood cells from healthy centenarians. Exp. Cell Res. 248, 234–242. Pacifici, R.E., Davies, K.J.A., 1991. Protein, lipid and DNA repair systems in oxidative stress: the free radical theory of aging revisited. Gerontology 37, 166 –180. Packer, L., Feuher, K., 1977. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 267, 423–425. Pani, G., Colavitti, R., Borrello, S., Galeotti, T., 2000. Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes. Biochem. J. 347 (1), 173– 181. Pawelec, G., Barnett, Y., Mariani, E., Solana, R., 2002. Human CD4 þ T cell clone longevity in tissue culture: lack of influence of donor age or cell origin. Exp. Gerontol. 37 (2/3), 265–269.
530
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
Tatla, S., Woodhead, V., Foreman, J.C., Chain, B.M., 1998. The role of reactive oxygen species in triggering proliferation and IL-2 secretion in T cells. Free Radic. Biol. Med. 26 (1/2), 14– 24. Schreck, R., Rieber, P., Baeuerle, P.A., 1992. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NFkappa B transcription factor and HIV-1. EMBO J. 10, 2247–2258.
Slagboom, P.E., Droog, S., Boomsma, D.I., 1994. Genetic determination of telomere size in humans: a twin study of three age groups. Am. J. Hum. Genet. 55, 876–882. von Zglinicki, T., Saretzki, G., Docke, W., Wolf-Lotze, C., 1995. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model of senescence. Exp. Cell Res. 220, 186 –193.
Effects of a reduced oxygen tension culture system on human T cell clones as a function of in vitro age Orla Duggana, Paul Hylanda, Kathryn Annetta, Robin Freeburna, Christopher Barnettb, Graham Pawelecc, Yvonne Barnetta,* a
Cancer and Ageing Research Group, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK b Research Support Office, Trent Building, University Park, University of Nottingham, Nottingham NG7 2RD, UK c University of Tu¨bingen, Tu¨bingen Ageing and Tumour Immunology Group (TATI), ZMF, Waldho¨rnlestr. 22, D-72072 Tu¨bingen, Germany Received 14 July 2003; received in revised form 26 November 2003; accepted 1 December 2003
Abstract Oxidative DNA damage has been suggested to contribute to the decline in T cell clone (TCC) function with increased age in vitro. To test this hypothesis the effect of a reduced oxygen tension culture system (6% O2) on TCCs was examined. Specifically, the effects of the altered culture conditions on DNA damage levels, in vitro lifespan and proliferative capacity were assessed in five independently derived human CD4 þ TCCs. DNA damage levels over the entire lifespan were significantly lowered by reducing oxygen tension. Lifespan (total population doublings (PDs) achieved) and proliferative capacity (PDs/week) were reduced for all clones under reduced oxygen tension when compared to standard culture conditions. This observed tendency warrants further investigation using a greater number of clones from donors of all age groups before definitive conclusions regarding the effect of low oxygen tension on the lifespan and proliferative capacity of TCC can be made. However, these results may suggest that the reduced oxygen tension culture system has interfered with some aspect of T cell biology not yet examined within the remit of this study. q 2004 Elsevier Inc. All rights reserved. Keywords: Reduced oxygen tension; T cell clones; Oxidative DNA damage; Lifespan; Proliferative capacity
1. Introduction T cells in vivo are exposed to reactive oxygen species (ROS) from both intrinsic (e.g. mitochondria, oxidases, peroxisomes) and extrinsic sources (e.g. radiation, pollution, xenobiotics) (Pacifici and Davies, 1991; Ames et al., 1993). At sites of inflammation, cells of the immune system are exposed to high levels of ROS, which are produced as part of a normal immune response. ROS produced in excess may cause damage to T cell biomolecules and so result in altered T cell function or loss. Previous investigations within our laboratory found that in ex vivo T cells an increase in levels of genetic damage as a function of age (King et al., 1994, 1997; Barnett and King, 1995) and in human T cell clones (TCCs) cultured under 20% O2 tension levels of oxidative DNA damage increased in TCCs with * Corresponding author. Tel.: þ44-28-7032-4163; fax: þ 44-28-70324965. E-mail address: [email protected] (Y. Barnett). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.12.010
increasing time in culture (Hyland et al., 2000, 2001). Apart from the potentially detrimental effects of cellular biomolecules, ROS are also important as intracellular signalling molecules within T cells (Kamata and Hirata, 1999; Schreck et al., 1992). In this study we have examined the effect of reduced oxygen tension culture conditions on human CD4 þ TCCs. Proliferative capacity, lifespan and levels of DNA damage were examined in the TCCs grown under reduced oxygen tension throughout their lifespan.
2. Materials and methods 2.1. Culture of peripheral blood derived human CD4 þ T cell clones Five independently derived human TCCs were examined. The 400 series clones were obtained from
526
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
a 26-year-old overtly healthy laboratory worker, the 385 series clones were derived from an overtly healthy 45-yearold adult donor, and the 399 series clones were derived from a healthy 80-year-old donor conforming to the SENIEUR protocol for healthily aged individuals. Clones were grown in a serum-free medium (2 ml per well), X-Vivo 15 (BioWhittaker), at concentrations of 4 £ 105 cells per well, along with 2 £ 105 gamma-irradiated (80 Gy) RJK853 cells per well (EBV-transformed B-lymphoblastoid cell line with complete hprt deletion, a gift from Dr Jane Cole, MRC Cell Mutation Unit, University of Sussex, UK), as feeder cells. Clones maintained in standard culture conditions were incubated at 37 8C in a 5% CO2, 21% O2, 74% N2 atmosphere in a 7-day cycle. On days 1 and 4 of the cycle the clones were supplemented with 400 U/ml recombinant human IL-2 (Aldesleukin, Chiron, UK). On day 7 of the cycle, the cells were harvested and a viable cell count was performed using an improved Neubauer Counting Chamber, and a new culture cycle was set up with fresh medium and RJK853 feeder cells. At the end of each 7-day growth cycle, samples were removed and cryopreserved in a medium made up of 10% DMSO, 20% foetal bovine serum and 70% X-Vivo 15 and stored in liquid nitrogen. 2.2. Reduced oxygen tension culture conditions Reduced oxygen tension culture conditions were achieved by incubating the clones at 37 8C in a 3% O2, 5% CO2, 92% N2 atmosphere for 10 weeks and then at 37 8C in a 6% O2, 5% CO2, 89% N2 atmosphere until the end of the lifespans of the clones. The TCCs were shifted to a higher oxygen tension after 10 weeks because there was no measurable proliferation at 3% O2 although the clones remained viable. 2.3. Determination of T cell clone lifespan and proliferative capacity TCC proliferative capacity was calculated by determining the number of population doublings (PDs) achieved by each clone, per week, using the formula: PD ¼ log2(cell count at end of cycle) 2 log2(number of cells at start of cycle) Lifespan for each TCC was determined by the total cumulative PDs achieved. 2.4. Determination of oxidative DNA damage levels in T cell clones Levels of oxidative DNA damage in TCCs grown were determined at three time points during their in vitro lifespans. Details of the modified alkaline comet assay used have been described previously (Collins et al., 1993; Hyland et al., 2000, 2001). The modified comet assay uses formamidopyrimidine glycosylase, which recognises
Table 1 Total population doublings achieved by TCCs maintained under different culture conditions Clone
Standard oxygen tension
Reduced oxygen tension
385-2 385-7 399-35 399-37 400-23 Mean lifespan (PD)
69.9 73.5 72.1 78.1 80.7 74.9 ^ 4.4
61.6 71.5 45.0 51.9 51.7 56.3 ^ 10.3*
*Significantly lower, p , 0:05:
oxidatively modified purines (Boiteux et al., 1992) and endonuclease III (Endo III) which recognises oxidatively modified pyrimidines (Asahara et al., 1989). These enzymes nick DNA at the sites of oxidatively damaged nucleotides, creating single strand-breaks, which can be detected with the assay. Comet analysis was performed using Komet 3.0 analysis software (Kinetic Imaging, UK), counting 50 cells per slide. DNA damage results were expressed as the percentage of DNA in the comet tail. 2.5. Statistical analysis Lifespan and proliferative capacity results were tested for significance using paired 2-sample 1-tailed Student’s t-tests assuming equal variance.
3. Results 3.1. T cell clone lifespan and proliferative capacity Table 1 shows the cumulative PDs achieved by each clone and the average lifespan achieved overall in each culture condition. The results in this table show that clones grown under the reduced oxygen conditions had shorter lifespans than their respective controls ðp , 0:05Þ: Reduced oxygen conditions had the greatest effect on lifespans in TCC from the young and old donors, whereas the TCC from the middle-aged donor were minimally effected. Table 2 details the mean proliferative Table 2 Mean proliferative capacity per week (PDs/week) for each T cell clone maintained under different culture conditions Clone
Standard oxygen tension
Reduced oxygen tension
385-2 385-7 399-35 399-37 400-23 Mean PD/week
1.9 ^ 0.6 2.1 ^ 0.7 2.1 ^ 0.9 2.2 ^ 0.8 2.1 ^ 1.0 2.1 ^ 0.1
1.1 ^ 0.6 1.2 ^ 0.6 0.8 ^ 0.5 0.9 ^ 0.6 0.9 ^ 0.6 1.0 ^ 0.2*
*Significantly lower, p , 0:05:
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
capacity per week for each TCC under each of the culture conditions. The results presented in Table 2 show that clones grown under reduced oxygen tension also had reduced replicative capacity compared to controls ðp , 0:05Þ:
527
3.2. Oxidative DNA damage levels in T cell clones Fig. 1 illustrates the levels of DNA damage as a function of in vitro age in TCCs grown under standard or reduced oxygen conditions. From this figure it can be seen
Fig. 1. Oxidative DNA damage levels in T cell clones maintained under standard and reduced oxygen tension conditions. (a) DNA strand breaks and alkali labile sites, (b) oxidatively damaged pyrimidine bases plus DNA strand breaks and alkali labile sites and (c) oxidatively damaged purine bases plus DNA strand breaks and alkali labile sites.
528
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
that throughout the lifespan of the clones, DNA damage levels in those cultured under reduced oxygen tension were maintained at lower levels when compared to the same clones grown under standard conditions. Fig. 1(a) shows the DNA strand breaks and alkali labile sites present throughout the lifespan of the TCCs under both culture conditions. Fig. 1(b) shows the DNA strand breaks and alkali labile sites plus oxidative DNA damage to pyrimidine bases and Fig. 1(c) shows the DNA strand breaks and alkali labile sites plus oxidative DNA damage to purine bases under both reduced and standard oxygen tensions. Fig. 1(b) and (c) show that the levels of oxidative DNA damage plus DNA strand breaks and alkali labile sites remain low throughout the lifespan of the TCCs cultured under reduced oxygen tension. In comparison, the control clones cultured under standard oxygen tension showed an increase in all aspects of oxidative DNA damage levels with in vitro age. Donor age appeared to exert no effect on the observed levels of DNA damage. This is in accordance with previously reported studies (Hyland et al., 2000, 2001).
4. Discussion The free radical theory of ageing (Harman, 1956) suggested an important role for ROS in the aetiology of ageing and age-related pathologies. Since this theory was first reported much evidence has been found to support it (Beckman and Ames, 1998). A number of investigations have indicated that there may be a relationship between oxygen tension (and thus ROS levels), cellular senescence and lifespan. The results of previous studies have suggested a beneficial effect of reduced oxygen tension on human diploid fibroblast (HDF) cultures. Packer and Feuher (1977) found that growing two types of HDFs (WI-37 and IMR-90) under 10% oxygen extended their lifespans (measured in total PDs) by 25%. More recently, Chen et al. (1995) have demonstrated that long-term culture of HDFs under a more physiological oxygen concentration of 3% O2 could significantly extend the in vitro lifespan of the fibroblasts. The HDFs grown under reduced oxygen tension attained approximately 50% more PDs during their in vitro lifespan. It was also noted that HDFs cultured under reduced oxygen tension had increased rates of proliferation compared to cells cultured under standard 20% O2. Chen et al. also reported that the reduced oxygen tension helped to maintain a juvenile HDF phenotype. The converse effect has also been demonstrated by von Zglinicki et al. (1995), where the replicative lifespan of HDFs were shortened under increased oxygen tension of 40% O2. The beneficial effect of a low oxygen tension environment is likely to be due to reduced free radical levels under such conditions, leading to lowered levels of radical induced biomolecule damage. In view of the fact that TCCs grown under standard (20% O2) culture conditions accumulate oxidative DNA
damage with age (Hyland et al., 2000, 2001), the potential beneficial effects of lowered oxygen tensions on TCCs were investigated. The results of the present study demonstrated that levels of oxidative DNA damage were maintained at a relatively low level throughout the lifespan of the clones grown under reduced oxygen tension. Standard TCC cultures exhibited an age-related increase in the levels of oxidative DNA damage. However, the TCC in this study had shorter lifespans and reduced replicative capacities under reduced oxygen tension when compared to the clones grown under standard culture conditions. This effect appeared to be greater in TCC from the young and old donor, and less in the clone from the middle-aged donor. This may not be significant as it has been established that TCC longevity is independent of donor age (McCarron et al., 1987; Pawelec et al., 2002). This is in agreement with recent work done on human fibroblasts, where the long-established view that fibroblast longevity in culture was dependent on donor age was shown not to be the case when donor health status and biopsy conditions were rigorously controlled (Cristofalo et al., 1998). However, this observed tendency towards impaired proliferation warrants further investigation using a greater number of clones from donors of all age groups before any definitive relationship between low oxygen tension culture systems, donor age, lifespan and proliferative capacity of TCC can be proposed. These observations on lifespan in TCC conflict with the previous HDF studies. There may be basic differences in the biology of ageing in fibroblasts and T cells. For example, recent studies have shown that telomere lengths in fibroblasts ex vivo are not decreased in the elderly, up to centenarian age, although the same fibroblasts did show telomere length decreases upon in vitro culture (Mondello et al., 1999). In contrast, telomere length in blood cells ex vivo was shown to be related to donor age (Slagboom et al., 1994), even in the same individuals mentioned above, whose fibroblasts failed to show a decrease, although there is a great deal of inter-individual variation (Mondello et al., 1999). In addition TCC are known to be more susceptible to variations in culture conditions than fibroblast cultures (Pawelec et al., 2002). Results from previous investigations on senescence and longevity were derived from polyclonal fibroblast populations, which behave differently from the monoclonal TCC populations. Not only do these two different cell types behave differently under similar conditions, but the fact that HDFs are polyclonal means that the studies are examining the behaviour of the longest-lived clones within that population. However, when using the TCC culture system the behaviour of each single cell type is investigated. It is possible that polyclonal T cell populations studied under these same conditions may behave in a similar manner to the HDFs, and this is currently under investigation within our laboratory.
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
It seems probable that some aspect of T cell biology not yet examined within the remit of this study may have been affected by the reduced oxygen tension culture conditions. This interference could explain the reasons for the reduced proliferation of the TCCs grown under reduced oxygen tensions, and may have contributed the reduced lifespans of these TCCs. Although ROS are typically thought of as harmful molecules, which cause cellular damage, they are also known to be important intracellular signalling molecules within T cells (Kamata and Hirata, 1999; Schreck et al., 1992). Examples are the requirement of ROS for cell proliferation induced by lectin, and the involvement of ROS in T cell signalling events such as protein tyrosine phosphorylation and the activation of JNK1 (Pani et al., 2000). Therefore one possible reason for the observed reduced proliferation in TCC may be that the 3/6% oxygen culture conditions may have resulted in a reduced intracellular level of oxygen free radicals and so may have altered the overall redox balance of the TCCs. The lack of an age-related increase in oxidative DNA damage levels in clones grown under reduced oxygen tension, may provide evidence of lower levels of oxygen-derived free radicals in these clones. In turn, lowered levels of oxygen-derived free radicals may result in an interference with signalling pathways, e.g. the redox-sensitive activation of transcription factors such as NFkB or AP-1, involved in T cell activation and proliferation (Tatla et al., 1998). This interference may have contributed to the reduced proliferation and reduced lifespans of the TCCs cultured under reduced oxygen tensions. The results of this study clearly warrant further investigation. We are currently investigating the effects of various concentrations of oxygen tension on T cell biology. Both monoclonal and polyclonal T cell populations are being included in these analyses. In addition, it is necessary to investigate other aspects of T cell biology such as signalling pathways effected by oxygen tension. The preliminary results of this study highlight that any interventions aimed at reducing the accumulation of oxidative damage with age in vivo in T cells (or other cell types) should be carefully optimised to ensure that normal cellular activities can still occur and that there are no other detrimental effects on other aspects of the target cell biology.
Acknowledgements The authors wish to acknowledge: University of Ulster for a Vice Chancellors Research Studentship to OD; The Department of Education and Learning for a Studentship to KA; The European Union for funding support under the aegis of Immunology and Ageing in Europe (ImAginE; QLK6-CT-1999-O2031) and T cell Immunity and Ageing (TCIA; QL1C6-CT-2002-02283).
529
References Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants and the degenerative diseases of aging. Proc. Natl Acad. Sci. USA 90, 7915–7922. Asahara, H., Wisort, P.M., Bank, J.F., Bakerian, R.H., Cunningham, R.P., 1989. Purification and characterisation of Escherichia coli endonuclease III from the cloned nth gene. Biochemistry 28, 4444–4449. Barnett, Y.A., King, C.M., 1995. An investigation of antioxidant status, DNA repair capacity and mutation as a function of age in humans. Mutat. Res. 338, 115 –128. Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 70 (2), 547– 581. Boiteux, S., Gajewski, E., Laval, J., Dizdaroglu, M., 1992. Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine–DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization. Biochemistry 31, 106– 110. Chen, Q., Fischer, A., Reagan, J.D., Yan, L.-J., Ames, B.N., 1995. Oxidative DNA damage and senescence of human diploid fibroblast cells. Proc. Natl Acad. Sci. USA 92, 4337–4341. Collins, A.R., Duthie, S.J., Dobson, V.L., 1993. Direct enzymatic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14, 1733–1735. Cristofalo, V.J., Allen, R.G., Pignolo, R.J., Martin, B.G., Beck, J.C., 1998. Relationship between donor age and the replicative lifespan of human cells in culture: a re-evaluation. Proc. Natl Acad. Sci. USA. 95, 10614–10619. Harman, D., 1956. Ageing: a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300. Hyland, P., Duggan, O., Hipkiss, A., Barnett, C., Barnett, Y., 2000. The effects of carnosine on oxidative DNA damage levels and in vitro lifespan in human peripheral blood derived CD4 þ T cell clones. Mech. Ageing Dev. 121, 203–215. Hyland, P., Barnett, C., Pawelec, G., Barnett, Y., 2001. Age-related accumulation of oxidative DNA damage and alterations in levels of p16INK4a/CDKN2a, p21WAF1/CIP1/SDI1 and p27KIP1 in human CD4 þ T cell clones in vitro. Mech. Ageing Dev. 122 (11), 1151–1167. Kamata, H., Hirata, H., 1999. Redox regulation of cellular signalling. Cell. Signal. 11 (1), 1–14. King, C.M., Gillespie, E.S., McKenna, P.G., Barnett, Y.A., 1994. An investigation of mutation as a function of age in humans. Mutat. Res.DNAging Genet. Instabil. Aging 316 (2), 79 –90. King, C.M., Bristow-Craig, H.E., Gillespie, E.S., Barnett, Y.A., 1997. In vivo antioxidant status, DNA damage, mutation and DNA repair capacity in cultured lymphocytes from healthy 75–80 year old humans. Mutat. Res. 377, 137 –147. McCarron, M., Osborne, Y., Story, C., Dempsey, J.L., Turner, R., Morley, A., 1987. Effect of age on lymphocyte proliferation. Mech. Ageing Dev. 41, 211–218. Mondello, C., Petropoulou, C., Monti, D., Gonos, E.S., Franceschi, C., Nuzzo, F., 1999. Telomere length in fibroblasts and blood cells from healthy centenarians. Exp. Cell Res. 248, 234–242. Pacifici, R.E., Davies, K.J.A., 1991. Protein, lipid and DNA repair systems in oxidative stress: the free radical theory of aging revisited. Gerontology 37, 166 –180. Packer, L., Feuher, K., 1977. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 267, 423–425. Pani, G., Colavitti, R., Borrello, S., Galeotti, T., 2000. Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes. Biochem. J. 347 (1), 173– 181. Pawelec, G., Barnett, Y., Mariani, E., Solana, R., 2002. Human CD4 þ T cell clone longevity in tissue culture: lack of influence of donor age or cell origin. Exp. Gerontol. 37 (2/3), 265–269.
530
O. Duggan et al. / Experimental Gerontology 39 (2004) 525–530
Tatla, S., Woodhead, V., Foreman, J.C., Chain, B.M., 1998. The role of reactive oxygen species in triggering proliferation and IL-2 secretion in T cells. Free Radic. Biol. Med. 26 (1/2), 14– 24. Schreck, R., Rieber, P., Baeuerle, P.A., 1992. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NFkappa B transcription factor and HIV-1. EMBO J. 10, 2247–2258.
Slagboom, P.E., Droog, S., Boomsma, D.I., 1994. Genetic determination of telomere size in humans: a twin study of three age groups. Am. J. Hum. Genet. 55, 876–882. von Zglinicki, T., Saretzki, G., Docke, W., Wolf-Lotze, C., 1995. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model of senescence. Exp. Cell Res. 220, 186 –193.