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Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland
Correspondence: Dr. Nan-ping Weng, Laboratory of Immunology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Box 21, Baltimore, MD 21224. E-mail: wengn{at}grc.nia.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONHEMATOPOIETIC STEM CELLSLYMPHOID LINEAGEMYELOID LINEAGECONCLUSIONREFERENCES |
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Key Words: lymphocyte • monocyte • granulocyte • mast cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONHEMATOPOIETIC STEM CELLSLYMPHOID LINEAGEMYELOID LINEAGECONCLUSIONREFERENCES |
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Each type of leukocyte has a distinct differentiation and maturation process. The myeloid progenitor cells differentiate in the BM to become granulocyte, monocyte, and mast cell and then migrate into the peripheral blood with a relative short lifespan (hours to days) [1 ]. Lymphoid progenitor cells also differentiate in BM. However, only B lymphocytes mature in BM [2 ], whereas T lymphocytes mature in the thymus [3 ]. Mature, naïve B and T lymphocytes have a much longer lifespan than myeloid-derived mature cells [4 ].
Mature leukocytes circulate in blood and exhibit characteristic patterns of growth, migration, and cellular function. Myeloid-derived, mature leukocytes are terminally differentiated cells and do not undergo further cell division. Mature lymphocytes, however, retain the ability for a rapid proliferation and differentiation, producing a large number of cells upon antigenic stimulation. Some activated lymphocytes become long-lived memory cells that are capable of further, extensive cell divisions upon reencounter of specific antigen. Thus, dependence on cellular replicative capability is one of the key differences in the function of myeloid- and lymphoid-derived cells.
The lifespan of cells may be regulated by multiple factors. Recently, telomeres and telomerase have been implicated for their roles in regulating replicative lifespan. Telomeres are special structures located at the end of eukaryotic chromosomes, which consist of an array of tandem heximer DNA repeats (TTAGGG)n and associated proteins. Constraints on the ability of DNA polymerase to completely replicate the extreme 3' end of chromosomes lead to a loss of telomere repeats and have been observed in human somatic cells with cell division [5 , 6 ]. It has also been suggested that telomeres can change from a normal "capped" state to an abnormal "uncapped" state [7 ]. The numbers of uncapped telomere ends increase in cells with critically short telomeres, which can signal cells to enter cell-cycle arrest or senescence. Thus, telomere length and its states serve as a biological regulator underlying the limited division potential of human cells.
Telomerase is a ribonucleoprotein enzyme that can synthesize telomeres and compensate for the cell division-associated loss of telomeres. Although not all the components of telomerase holoenzyme have been identified, the core components of telomerase consist of telomerase-reverse transcriptase [8 , 9 ] and telomerase RNA template [10 ]. The expression of telomerase is highly regulated during development and differentiation in a tissue-specific manner. Many tissues express telomerase only during development and differentiation, but lose expression in terminally differentiated or mature cells. However, germ cells and some stem cells retain expression of telomerase. This phenomenon has been attributed to the fact that most terminally differentiated cells do not require substantial cell divisions for their function and that unregulated telomerase may facilitate the transformation of normal cells. Recent studies showed that telomerase is expressed in stem cells [11 , 12 ], stem-like cells (such as basal-layer cells in the skin and intestine-endothelial cells) [13 , 14 ], and lymphocytes [15 , 16 ]. The questions of how telomere length is regulated in cells that express telomerase and whether telomerase expression extends the replicative lifespan of these cells remain controversial.
Age associated with decline of the overall cellular function has been demonstrated in innate and adaptive immune responses [17 18 19 ]. Changes with aging include a decrease in production of hematopoietic stem cells [20 ], clonal or oligoclonal expansion of CD28- T cells [21 ], and intrinsic defects in cellular function of leukocytes [22 ]. Because the function of the immune system is highly dependent on the capacity for extensive cell division and clonal expansion of lymphocytes, it is of great interest to understand if age-related processes result in a loss of the replicative capacity of lymphocytes. In this review, I will summarize the recent studies on telomere length and telomerase expression in human leukocytes during differentiation and aging.
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HEMATOPOIETIC STEM CELLS |
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TOPABSTRACTINTRODUCTIONHEMATOPOIETIC STEM CELLSLYMPHOID LINEAGEMYELOID LINEAGECONCLUSIONREFERENCES |
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There is an accumulating body of data derived from comparative analyses of telomere length of leukocytes between donor and recipient after BM transplantation (BMT) with hematopoietic stem cells [27 28 29 30 31 32 33 34 ]. To reconstitute the hematopoietic systems in the recipients, the donor hematopoietic stem cells must undergo extensive cell divisions to generate a sufficient number of cells. Notaro et al. [27 ] compared telomere length in granulocytes between donor and recipient of 11 BMT pairs and found a significant loss of telomere length in the recipients. Wynn et al. [28 , 29 ] compared telomere length in leukocyte, neutrophil, and T cells of 28 BMT cases and also found loss of telomeres in the recipients. Although different types of leukocytes were analyzed in the different studies, various degrees of telomere shortening were observed in all studies. Together, these results suggest that unlike germ cells, hematopoietic stem cells appear to lose telomere length with cell division and age (Fig. 1 ).
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LYMPHOID LINEAGE |
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TOPABSTRACTINTRODUCTIONHEMATOPOIETIC STEM CELLSLYMPHOID LINEAGEMYELOID LINEAGECONCLUSIONREFERENCES |
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T lymphocytes
Telomere shortening has been found in blood CD4+ and
CD8+ T cells with age in vivo [37
,
38
, 40
] and with cell division in vitro
[37
]. Son et al. [38
] demonstrated a
cross-sectional analysis of CD4+ T cells of 121 normal
donors aged from newborn to 94 years and showed that the rates of
telomere shortening of CD4+ and CD8+ T cells
are 35 and 26 bp/year, respectively. Although telomere loss appears to
continue through life, Rufer et al. [40
] found that
there was rapid loss of telomeres in the first year of life at a rate
that is 30-fold higher than that in the subsequent nine decades.
Recently, Friedrich et al. [41
] showed changes in
telomere length of leukocytes during human fetal development. They
found a rapid and significant decline in mean telomeric restriction
fragment length between 27 and 32 weeks of gestation
(P=0.02, r=0.79), followed by a period of no
significant loss of telomere repeats between 33 and 42 weeks of
gestation. In addition to telomere loss under normal development and
aging, an accelerated loss of telomeres was observed under an abnormal
condition in vivo. T cells from Down syndrome patients showed a
significantly higher rate of telomere loss with donor age (133±15
bp/year) compared with age-matched controls (41±7.7 bp/year;
P<0.0005) [42
].
Because the length of telomeres reflects past cell division of a cell, it may be useful for assessing the potential precursor and descendent relationship within a single lineage of cells. The first such analysis was done in naïve and memory CD4+ T cells from a group of 20 donors aged from 25–70 years [37 ]. It was found that telomere length measured by the mean terminal restriction fragments (TRF) length was 1.4 kb longer in naive CD4+ T cells than in memory T cells from the same donors and that this difference remained constant over a wide range of donor ages [37 ]. The rate of loss of telomeres with age was similar between naïve and memory CD4+ T cells (approximately 33 bp/year). A subsequent, more extensive analysis by Rufer et al. [40 ], using a much larger cohort (over 500 donors aged from 0–90 years), confirmed that naïve CD4+ T cells have consistently longer mean telomere length than memory CD4+ T cells. The rate of telomere loss in that study, however, was slightly greater in memory CD4+ T cells (51 bp/year) than in naïve CD4 T cells (39 bp/year) with age. Burns et al. [43 ] extended the telomere length analysis to memory T cells responding to tetanus toxoid and Candida albicans. They found that Candida- or tetanus-reactive memory T-cell populations demonstrated a significant reduction of telomere length even when compared with the phenotypically defined memory CD45RO+ T-cell populations isolated from peripheral blood mononuclear cells (PBMC) [43 ].
Similar findings of telomere length differences were also observed in CD8+ T-cell subsets. Monteiro et al. [44 ] showed that CD8+CD28- T cells have shorter telomeres compared with CD8+CD28+ T cells [45 ]. Rufer et al. [40 ] also showed that naïve CD8+ T cells have longer telomeres than memory CD8+ T cells. These findings suggest that the differentiation of memory cells from naive precursors occurs with substantial cell division. Thus, telomere length could be a useful marker for determining the precursor/descendent relationship within a cell lineage.
The cross-sectional analysis of age-associated telomere shortening implies that accumulating cell divisions in vivo with age may be the cause for the telomere loss. A more direct assessment of such a relationship was carried out by culturing primary T cells over 3–4 months in vitro. Cell division was estimated by the calculation of mean population doublings (MPD) in parallel with the measurement of telomere length changes. Analysis of long-term, cultured naïve and memory CD4+ T cells found that naïve and memory CD4+ T cells lose telomeres during the course of long-term culture. The rate of telomere loss per cell division ranged from 50–100 bp/year in naive and memory CD4+ T cells. It is interesting that naive CD4+ T cells that have longer telomeres undergo a greater number of MPD than memory CD4+ T cells [37 ]. Similarly, long-term, cultured CD8+ T cells also lost telomere length with cell divisions [46 ]. Thus, CD4+ and CD8+ T cells appear to lose telomere length with cell division in vitro. However, it is not clear whether the cease of cell division in the end of long-term culture results from an exhaustion of the division potential or lack of proper growth conditions. Therefore, longitudinal analyses of lymphocytes ex vivo and in vitro with optimized culture conditions will be necessary in order to understand the mechanisms of telomere length regulation and its role in lymphocyte replicative lifespans.
Considering the importance of cellular replicative capability in lymphocyte function, it is conceivable that some telomere protection mechanisms may work in lymphocytes. The first evidence that lymphocytes might express telomerase was from stuides by Broccoli et al. and others [15 , 16 ]. Both showed that a low level of telomerase was detected in peripheral blood lymphocytes isolated from normal donors. Subsequent analysis found that telomerase activity is highly regulated during T-cell development and differentiation. High levels of telomerase were detected in thymocytes [39 ] and low-to-undetectable levels in mature CD4+ and CD8+ T cells. It is intriguing that telomerase activity can be induced in these mature CD4+ and CD8+ T cells after in vivo antigenic stimulation or in vitro activation [39 , 47 , 48 ]. These findings indicate that human telomerase is not restricted to immortal or germ cells and specifically raise questions of the functional role of telomerase in lymphocytes and of aging influence in telomerase expression.
Although the precise role of telomerase in lymphocyte function remains to be elucidated, several studies showed that the levels of telomerase activity in lymphocytes appear to correlate with the telomere states in cultured lymphocytes [46 , 49 ]. Studies of long-term cultured CD4+ and CD8+ T cells show that telomere length was stable when telomerase activity was high, whereas telomere length shortened when telomerase activity was low-to-undetectable. Recent longitudinal analyses of cytotoxic CD8+ T-cell responses against Epstein-Barr virus (EBV) in vivo show that EBV-specific CD8+ T cells in patients with acute infectious mononucleosis (AIM) undergo considerable expansion without telomere length loss and that these cells express high levels of telomerase activity [50 , 51 ]. Thus, it appears that there is a quantitative relationship between telomerase activity and maintenance of telomere length in lymphocytes. Understanding how telomerase is regulated will undoubtedly help to elucidate telomere length dynamics in lymphocytes and may provide new means of immunomodulation for clinical intervention.
To determine whether age affects telomerase expression in lymphocytes, Son et al. [38 ] recently demonstrated a cross-sectional analysis of telomerase induction. They showed that age did not appear to alter the magnitude of telomerase activity induced after stimulation of CD4+ and CD8+ T lymphocytes through T-cell receptor (TCR) and costimulatory receptors or in response to phorbol 12-myristate 13-acetate (PMA) plus ionomycin treatment in a 3-day culture [38 ]. These findings suggest that the capacity for telomerase expression may be stable and does not change with age in CD4+ and CD8+ T lymphocytes. It remains to be determined, however, whether sustained induction of telomerase activity changes with age.
B lymphocytes
Despite the many similarities between T and B lymphocytes, the
latter display some unique features in regulation of telomere length
and telomerase activity. Like T lymphocytes, B lymphocytes lose
telomeres with age, but the rate of telomere attrition appears to be
slower than T cells (19 bp/year for B cells compared with 30–50
bp/year for T cells) [38
]. Although there is no
available method for growing normal B lymphocytes for the long term, a
study using EBV-immortalized B cells showed that telomere shortening
occurs with cell division in these B cells in vitro [8
].
One striking difference between T and B lymphocytes is in relative
telomere lengths in their subsets. Unlike naïve and memory T
cells, there is no obvious telomere shortening in memory B cells
compared with naïve B cells isolated from tonsil. In contrast,
germinal center (GC) B cells as the descendent of naïve B cells
have significantly longer telomeres than naïve or memory B
cells. Although there is no study of telomere length in peripheral
blood naïve and memory B cells to directly compare with T
cells, these findings suggest that regulation of telomere length in B
lymphocytes may differ from T lymphocytes.
Like T lymphocytes, telomerase activity was lower-to-undetectable in resting, mature B lymphocytes freshly isolated from peripheral blood than was induced upon activation through B cell receptor, by superantigen, or by PMA/ionomycin [52 53 54 ]. An in vivo example of telomerase regulation in response to B-cell activation is found in GC B cells that have presumably differentiated from naïve B cells after antigenic activation and that express high levels of telomerase activity [53 ]. Although induction of telomerase activity in resting lymphocytes has been correlated with antigen receptor activation-induced cellular proliferation, Hu et al. [54 ] showed that proliferation of B lymphocytes induced by cytokines did not activate telomerase. Thus, it appears that the events of telomerase activation and cellular proliferation can be regulated independently. In addition, age does not appear to alter the magnitude of telomerase activity induced after in vitro stimulation of B lymphocytes through Ag and costimulatory receptors or in response to PMA plus ionomycin treatment [38 ].
The relationship of telomerase expression and telomere loss in B lymphocytes has been shown during B-cell differentiation [52 ]. In contrast to previous observations of telomere attrition during the process of somatic cell differentiation and cell division, GC B cells had significantly longer telomeres than those of precursor naive B cells, which were correlated with high levels of telomerase expression. These results suggest that telomerase may not only preserve but also lengthen telomeres in actively dividing GC B cells. Thus, telomerase may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.
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MYELOID LINEAGE |
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TOPABSTRACTINTRODUCTIONHEMATOPOIETIC STEM CELLSLYMPHOID LINEAGEMYELOID LINEAGECONCLUSIONREFERENCES |
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Granulocytes
Granulocytes are a group of leukocytes that include neutrophils,
basophils, and eosinophils. Unlike lymphocytes, mature granulocytes do
not undergo cell division; therefore, telomere length of granulocytes
reflects myeloid progenitor cells. Rufer et al. [40
]
described an analysis of 301 normal donors aged from 0–90 years and
found that an overall loss of telomere length in granulocyte with age
is approximately 39 bp/year. Like T lymphocytes, granulocytes also
display a rapid loss of telomere length in the first year of life
[40
, 55
]. It is interesting that the
difference in telomere length between granulocytes and T lymphocytes in
peripheral blood appears to be influenced by age [29
,
40
]. Telomeres were generally shorter in granulocytes
than in T lymphocytes from young donors, whereas telomere was longer in
granulocytes than in T lymphocytes in aged individuals. This reversal
of telomere length between granulocytes and T lymphocytes with age may
reflect a significant increase of memory T lymphocytes that have
shorter telomeres than those in naïve T lymphocytes in the
elderly.
Telomerase expression in granulocytes is controversial. One study suggested that there was a low level of telomerase activity in the granulocyte fraction from peripheral blood [15 ]. However, subsequent studies failed to detect telomerase activity in granulocytes [56 57 58 59 ]. It is possible that a residual contamination of stem cells or lymphocytes in the granulocyte fraction contributes to the low activity of telomerase. Physiologically, it seems unlikely that mature granulocytes, which have a very short life and do not undergo subsequent cell divisions, need telomerase for their function.
Monocytes
There have been no studies of direct assessment of telomere length
changes in monocytes with age. Several studies using PBMC consisting of
10–15% monocytes and 60–70% lymphocytes showed telomere shortening
with age at a rate comparable with that of purified lymphocytes
[38
, 40
, 60
]. Like
granulocytes, mature monocytes do not undergo further cell division
after activation. Thus, the changes of telomere length in monocytes
with age are likely reflecting the changes of telomere length in
hematopoietic progenitor cells.
Mature monocytes do not express telomerase, but myeloid progenitor cells do appear to express telomerase. Studies of differentiation of a promyelocytic leukemia cell line (HL-60) in vitro demonstrated that telomerase is highly expressed in undifferentiated HL-60 cells but down-regulated after induced differentiation to monocytes or granulocytes [56 57 58 59 , 61 ]. The down-regulation of telomerase activity appears to be a differentiation process and is independent of cellular proliferation. These findings provide a direct link between telomerase activity and terminal differentiation and thus present a useful model to study regulation of telomerase activity in myeloid cells.
Mast cells
Like monocytes, no direct assessment of telomere length of mast
cells with age has been shown, and no telomerase activity was detected
in mature mast cells [62
]. However, telomerase is
expressed and regulated during mast-cell development from human
hematopoietic pluripotent cells (CD34+) and mast-cell
progenitor cells
(CD34+/CD117+/CD13+)
[62
]. It has been shown that a rapid increase in
telomerase activity preceded proliferation of hematopoietic stem- and
mast-cell progenitors in the presence of stem-cell factor and IL-3 or
IL-6. The induction of telomerase was transient, and telomerase
activity declined to basal levels before the appearance of mature mast
cells. These findings suggest that the transient induction of
telomerase activity is dependent on growth factor-mediated signals in
progenitor mast cells. The precise role of telomerase expression in
progenitor mast-cell differentiation requires further study.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONHEMATOPOIETIC STEM CELLSLYMPHOID LINEAGEMYELOID LINEAGECONCLUSIONREFERENCES |
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ACKNOWLEDGEMENTS |
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Received September 14, 2001; accepted September 18, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONHEMATOPOIETIC STEM CELLSLYMPHOID LINEAGEMYELOID LINEAGECONCLUSIONREFERENCES |
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