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Originally published online as doi:10.1189/jlb.0103014 on May 22, 2003

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(Journal of Leukocyte Biology. 2003;74:270-276.)
© 2003 by Society for Leukocyte Biology

Dramatic increase of telomerase activity during dendritic cell differentiation and maturation

Lin Ping, Azusa Asai, Aki Okada, Kenichi Isobe and Hideo Nakajima

Department of Basic Gerontology, National Institute for Longevity Sciences, Obu, Japan

Correspondence: Hideo Nakajima, M.D., Ph.D., Department of Basic Gerontology, National Institute for Longevity Sciences, Gengo36-3, Morioka-cho, Obu, Aichi 474-8522, Japan. E-mail: hideonak{at}nils.go.jp


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ABSTRACT
 
Telomerase, the reverse transcriptase that maintains telomere DNA, is usually undetectable in most adult tissues but is positive in embryonic tissues and in cancers. In addition, freshly islolated or in vitro-activated lymphocytes were shown to express high levels of telomerase activity, although its expression in myeloid cells including dendritic cells (DCs) is largely unknown. Here, we investigated telomerase activity during the differentiation and maturation process of DCs. In vitro culture of bone marrow (BM) cells with granulocyte macrophage-colony stimulating factor and interleukin-4 induced a dramatic increase of telomerase activity accompanied with their differentiation into DCs. Furthermore, stimulation with microbial components such as lipopolysaccharide (LPS), which triggers maturation of DCs, augmented the activity. In vivo responses of telomerase activity were also observed in splenic DCs by injection of LPS intraperitoneally. It is interesting that in old mice, telomerase activity of splenic DCs was significantly higher than young mice but rather decreased after LPS stimulation. By measuring expression of cell-surface activation markers, splenic DCs of old mice responded poorly to LPS stimulation. Such poor responses to LPS were also observed in BM-derived DCs. These different features of DCs between young and old mice may contribute to a pathogenesis to microbial infections.

Key Words: DC • aging • telomere


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INTRODUCTION
 
Dendritic cells (DCs) are professional antigen presenting cells (APCs) and play a critical role in primary immune responses [1 , 2 ]. DCs reside in an immature state in nonlymphoid tissues, where they efficiently capture and process antigens. Upon activation, they initiate a complex maturation process, which results in decreased antigen-processing capacities, enhanced expression of major histocompatibility complex (MHC) and costimulatory molecules, and migration into secondary lymphoid organs to prime acquired immune responses [3 4 5 6 ]. The maturation process is central to the function of DCs and enables them to perform different, highly specialized actions sequentially. There are many stimuli that can initiate this maturation process in vivo and in vitro. In fact, DCs are sensitive to many different indicators of infection, reflecting the key role to recognize a variety of pathogens. Inflammatory products and microbial components such as lipopolysaccharide (LPS), bacterial DNA [CpG-oligodeoxynucleotide (ODN)], and a synthetic dsRNA (poly I:C), which is often used as a model of viral infection, are all able to stimulate DCs to become activated and matured, professional APCs [7 8 9 10 ].

The ends of linear eukaryotic chromosomes are capped by specialized DNA–protein structures, called telomeres, which are composed of hexanucleotide repeats (TTAGGG)n [11 , 12 ]. Telomeric DNA is lost every time somatic cells divide, and such shortening may act as a mitotic clock, regulating the number of cell divisions. When telomeres are shortened to such a critical point that they may no longer stabilize chromosome ends, most of the cells exit from the cell cycle and die [13 14 15 ]. Telomerase is a ribonucleoprotein enzyme that is able to add telomeric repeats to chromosome ends. In the presence of telomerase, telomere lengths are extended or maintained, and replicative senescence is avoided [16 , 17 ]. In initial analysis, telomerase activity was only detectable in the early, immature cells, such as hematopoietic precursor cells in the bone marrow (BM) at infantile ages, but recent studies have demonstrated that it can be detected in normal, somatic cells. It has been shown that telomerase activity is up-regulated during T cell activation [18 19 20 21 ] and B cell differentiation [22 ], and high levels of telomerase activity were observed in thymocytes and germinal center B cells. However, a limited number of studies have examined the expression of telomerase activity in mouse tissues and reported low levels of activity in a variety of mouse tissues.

Here, we analyzed telomerase activity of DCs at various differentiation stages. A significant increase of telomerase activity was observed during the differentiation and maturation process of DCs, which was led in vitro and in vivo. We also measured telomerase activity of DCs with or without stimulation by microbial components and made a comparison between young and old mice.


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MATERIALS AND METHODS
 
Mice
C57BL/6J male mice were purchased from Chubu-Kagaku Laboratory (Aichi, Japan) and were kept under specific, pathogen-free conditions at our animal facilities. They were used between 6 and 10 weeks of age (young mice). C57BL/6J male mice aged 2 years (old mice) and 1 year (adult mice) were obtained from our animal center.

Reagents
LPS was purchased from Sigma Chemical Co. (St. Louis, MO) and was used at 200 ng/ml for stimulation of cultured cell. CpG-ODN, the oligonucleotide 1668 containing a "CpG-motif" marked with bold letters (5'-TCC-ATG-ACG-TTC-CTG-ATG-CT), was phosphorothioate-stabilized and synthesized by Hokkaido System Science Co. (Hokkaido, Japan) and was used at 1 µM in culture. Poly I:C was purchased from Sigma Chemical Co. and was used at 25 µg/ml for stimulation. Mouse granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 were purchased from eBioscience (San Diego, CA) and were used at 20 ng/ml and 10 ng/ml, respectively, in DC culture.

Generation of DC from BM cells and stimulation in vitro
BM-derived DCs (BM DCs) were generated by a modification of the protocol as described previously [23 ]. Briefly, BM cells were collected from tibias and femurs of C57BL/6J mice and passed through a nylon mesh to remove small pieces. After an incubation period of one-half day, nonadherent cells were removed by washing, and the plastic-adherent cells were cultured in RPMI 1640 (Sigma Chemical Co.) containing GM-CSF and IL-4 with 10% fetal calf serum. Half of the medium was exchanged with fresh medium every 2 days. At day 6, loosely adherent cells [immature DCs (imDCs)] were harvested by gentle pipetting, plated at 5 x 105 cells/ml, and cultured with LPS, CpG-ODN, or poly I:C for an additional 2 days to generate mature DCs (mDCs).

Measurement of telomerase activity
Harvested cells were washed twice with ice-cold phosphate-buffered saline (PBS). The pellets were resuspended and incubated with lysis buffer of the telomerase polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics, Mannheim, Germany) on ice for 30 min and then centrifuged for 20 min at 10,000 g at 4°C, and the supernatant was collected as an extract of telomerase. Then, telomerase activity of extracts was measured by the telomerase PCR-ELISA, according to the manufacturer’s instructions or a modified version of the standard telomerase repeat amplification protocol (TRAP). In brief, the cell extract (representing 1x103 cells) was incubated for 30 min at 25°C in a mixture containing the reaction mixture of the telomerase PCR-ELISA kit and was heated at 94°C for 5 min and then subjected to 40 cycles of PCR amplification (94°C for 30 s, 58°C for 30 s, and 72°C for 90 s). PCR products were detected by the photometric enzyme immunoassay, and the Microtiter plate reader (Bio-Rad, Hercules, CA) measured the absorbance of samples at 450 nm. All assays were performed in triplicate. In the telomerase PCR-ELISA, the level of telomerase activity in positive-control cell extract supplied in the kit was set to 100%, and the relative specific telomerase activity (RTA) of each extract was expressed as a percentage of the positive-control standard (mean±SD). In the TRAP assay, the cell extracts were subjected to PCR with 2 µCi {alpha}-32P-dCTP, and after the reaction, direct visualization of the TRAP ladder is possible on a 12.5% nondenaturing polyacrylamide gel electrophoresis, followed by autoradiography.

Flow cytometry
Expression of cell-surface molecules was analyzed by flow cytometry with standard procedures. Anti-CD11c (HL3), anti-CD40 (3/23), anti-CD86 (GL1), anti-I-A/I-E (M5/114.15.2), and an isotype-control antibody (Ab) were purchased from BD PharMingen (Los Angeles, CA). Dead cells were excluded by staining with propidium iodide, and data were analyzed by Cell Quest software.

LPS stimulation in vivo and isolation of splenic cells and peritoneal macrophages
LPS (10 µg) in PBS or only PBS was injected into mice intraperitoneally (i.p.). Every group has five mice, and three independent experiments were performed. Twenty-four hours later, spleens were dissected, and single-cell suspensions were prepared by passing through nylon mesh. Cells were separated by leukocyte separation medium (ICN Biomedicals, Aurora, OH). The low-density fraction was collected and incubated on ice with anti-CD11c microbeads (N418), anti-CD11b microbeads (M1/70.15.11.5), anti-CD90 microbeads (30-H12), or anti-CD19 microbeads (1D3; all microbeads were obtained from Miltenyi Biotec, Gladbach, Germany). An automated magnetic cell sorter (Miltenyi Biotec) sorted the positive cells, according to the manufacturer’s instruction. Peritoneal macrophages were collected by recovering attached cells after 3 h incubation of peritoneal exudates from i.p.-injected PBS. For in vitro stimulation, splenic DCs were cultured with 200 ng/ml LPS in the presence of GM-CSF and IL-4 for 48 h. Purified cells were assessed by flow cytometry and were subjected to telomerase activity measurement.


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RESULTS
 
Telomerase activity is up-regulated during in vitro differentiation of DCs
To address whether telomerase activity has changed during differentiation of DCs, in the first place, we took the well-performed and standardized method to induce DCs in vitro. ImDCs were generated from BM cells of 6- to 8-week-old mice by culturing with GM-CSF and IL-4, and telomerase activity and surface phenotype were checked every 2 days (Fig. 1A ). Telomerase activity of cells started to increase immediately after they were put in culture and then showed a sharp increase and reached the maximum level at day 8. Simultaneously, the ratio of CD11c-positive cells indicating differentiation to DCs was also increased in a similar manner (Fig. 1A) .



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  		Figure 1. Induction of telomerase activity during DC differentiation and maturation. (A) BM cells were isolated and cultured in medium containing GM-CSF and IL-4. The cells were collected every 2 days from days 0 to 10, and ELISA measured telomerase activity. The same cells were stained with anti-CD11c Ab and measured in flow cytometry. Subtracted by an isotype-control Ab staining, the percentage of the CD11c+ cell was calculated from whole events. (B) At days 0 and 6, nonadherent cells were collected and cultured for an additional 2 days with or without LPS and measured telomerase activity. (C) Telomerase activity was visualized by TRAP. The PCR products from BM (day 0), imDCs and mDCs (day 8) were subjected to electrophoretic gel. (D) At day 6 of BM culture, cells were harvested and incubated with or without CpG-ODN, poly I:C, or LPS for another 2 days. Telomerase activity of imDCs without stimulation and mDCs with stimulation was compared with TRAP. P, positive control (telomerase activity in positive-control cell extract supplied); N, negative control (telomerase activity measured without cell extract).

Exposure to microbial components such as LPS can induce mDC, characterized by up-regulation of costimulatory molecules. To assess the effect of LPS, we measured the telomerase activity of cells at several time points of culture with or without LPS stimulation. Although we could not see a LPS effect on telomerase activity at day 2 when BM cells were not well differentiated to DCs yet, telomerase activity of cells at day 8 stimulated with LPS (mDCs) was significantly higher than unstimulated ones (imDCs), expressing a middle level of telomerase activity (Fig. 1B) . The same results were observed in the TRAP assay (Fig. 1C) . To confirm the correlation between telomerase activity and DC maturation, we used other microbial origin inflammatory agents to activate imDCs. Recent studies have shown that LPS, CpG, and dsRNA are capable of triggering maturation of DCs via Toll-like receptor (TLR) signaling [10 , 24 ]. Our experiments showed CpG and poly I:C were also able to increase telomerase activity of imDCs comparably with LPS (Fig. 1D) . These results proved that telomerase activity of imDCs was indeed up-regulated during the maturation process by infectious agents. We obtained similar results in six independent experiments.

LPS can enhance telomerase activity of splenic DCs and peritoneal macrophages in vivo
LPS has also been shown to lead the maturation of DCs in vivo, and injection of mice with LPS can induce up-regulation of costimulatory molecule expression on splenic DCs [25 ]. To further explore the correlation between regulation of telomerase activity and stimulation of DCs, we investigated effects of LPS in vivo. Splenic cells from 10-week-old mice injected with LPS or only PBS were sorted by antibody-coated microbeads: CD11c beads for DCs, CD11b for monocytes (macrophages), CD90 for T cells, and CD19 for B cells. After sorting, cells were checked by flow cytometry and were shown to have over 90% purity (data not shown). Analysis of telomerase activity in splenic cells injected with only PBS revealed that myeloid lineage cells such as DCs and monocytes have middle or low levels of telomerase activity, and lymphoid cells (B cells and T cells) have relatively high levels of activity. However, by injection of LPS, the telomerase activity of DCs was enhanced dramatically, whereas an increase in other hematopoietic cells was only modest (Fig. 2A ). Together, these findings clearly indicate that LPS can also induce up-regulation of telomerase activity on DCs in vivo, and DCs are the potent responding cells to LPS stimulation in the spleen. Among CD11b+ cells sorted, ~25% cells also expressed CD11c that represented DCs. To investigate responses of other myeloid cells to LPS, we also measured telomerase activity of peritoneal macrophages (Fig. 2B) . Similar to DCs, peritoneal macrophages expressed the middle level of telomerase activity (~50% RTA) without stimulation and rose up to ~70% RTA after LPS stimulation in vivo.



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  		Figure 2. Effect of LPS on telomerase activities of splenic cells and peritoneal macrophages. (A) LPS or PBS was injected i.p. into mice. After 48 h, splenic cells were sorted by antibody microbeads and separated to DCs by anti-CD11c, monocytes (Mo) by anti-CD11b, T cells by anti-CD90, and B cells by anti-CD19. ELISA and TRAP measured telomerase activity of each population. P, positive control; N, negative control. (B) After LPS or PBS injection, ELISA measured telomerase activity of peritoneal macrophages (M).

Different features of DCs between young and old mice
To investigate the effect of age on changes of telomerase activity, we compared telomerase activity of purified splenic cells between young and old mice. Although telomerase activity of lymphoid cells (B and T cells) was mostly comparable, that of DCs in old mice was significantly higher than young mice (P<0.01; Fig. 3A ). Furthermore, splenic DCs from old mice rather decreased the telomerase activity after in vitro stimulation with LPS, although those from young mice showed a slight increase of ~10%. By FACS analysis, freshly isolated, splenic DCs from both generations showed similar cell-surface phenotype determined by CD11c, MHC class II, and CD86 expression (Fig. 3B) . However after stimulation, expression of activation markers such as MHC class II and CD86 was apparently lower in old mice, indicating poor responses to LPS (Fig. 3B) . We also tried in vivo stimulation by injection of LPS, but old mice were not resistant and died (data not shown).



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  		Figure 3. Comparison of telomerase activity and surface phenotye of splenic DCs between young and old mice. (A) Telomerase activities of freshly isolated splenic B cells, T cells, DCs, and LPS-stimulated DCs were compared between young and old mice. (B) Expression of CD11c and activation markers of DCs [MHC class II (I-A) and CD86] was measured by fluorescein-activated cell sorter (FACS) in freshly isolated splenic DCs and LPS-stimulated DCs from young (thin line) and old (bold line) mice. Background staining with irrelevant Ab was expressed by the dotted line.

Next, we compared the telomerase activities of BM DCs (Fig. 4 ). We generated imDCs in the same set of in vitro cultures of BM cells from young mice (6 week), adult mice (1 year), and old mice (2 years). Contrary to the results of splenic DCs, BM DCs from old mice had consistently lower telomerase activity than those from young and adult mice, both of which had similar activity (Fig. 4A) . At day 6, imDCs from each generation were stimulated with LPS, CpG-ODN, or poly I:C to induce maturation. In this experiment, up-regulation of telomerase activity after stimulation was observed in all generations, but still, the lowest activity in old mice was consistent (Fig. 4B) . By FACS analysis, expression of activation markers such as CD40, CD86, and MHC class II was lower in old mice, and this tendency became more significant after stimulation (Fig. 4C) . Thus, lower telomerase activity of BM DCs in old mice seemed associated with insufficient activation.



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  		Figure 4. Comparison of BM DCs among young, adult, and old mice. (A) BM cells from old, adult, and young mice were cultured in medium with GM-CSF and IL-4. Every 2 days, telomerase activity was analyzed as described in the legend to Figure 1 . (B) ELISA measured telomerase activity of DCs from each generation, treated or untreated with LPS, CpG, or poly I:C. (C) imDCs (thin line) and LPS-induced mDCs (bold line) from each generation measured expression of activation markers of DCs. I-A, MHC class II.


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DISCUSSION
 
Telomerase in DCs
Telomere has an essential role in stabilizing chromosome ends and in preventing end-to-end fusions, and its expression has been found to correlate with cell proliferation in many different types of cells. Thus, telomerase activation is important in determining the proliferative capacity of cells and counteracts telomere loss. Telomerase activity has been shown to increase upon activation of T and B cells; however, a limited number of studies showed the telomere length and telomerase activity in myeloid-lineage cells. It was reported that telomerase activity was up-regulated in mature myeloid progenitors by plating in cultures supplemented with IL-3, Flt3-ligand, and stem-cell factor, and telomerase activity was also induced during development of human mast cells from peripheral blood CD34+ cells [26 , 27 ]. GM-CSF was shown to suppress the telomerase activity and telomerase reverse transcriptase (TERT) expression by synergistic effect with retinoic acid in myeloid leukemia cells [28 ]. Thus, it raises the question as to whether DCs change telomerase activity during differentiation as a professional APC.

Here, we clearly demonstrated that telomerase activity is largely increased during the differentiation and maturation process of DCs. In in vitro induction of DCs with GM-CSF and IL-4, telomerase activity was low at the beginning in the whole extract from BM cells but gradually increased during differentiation into CD11c+ imDCs (Fig. 1) . When LPS, CpG-ODN, and dsRNA induced DC maturation, telomerase activity of imDCs, having a moderate level, rose to the peak after maturation. All microbial products we used for DC maturation transduced activation signals through the TLR pathway. TLRs used the same signaling molecules including MyD88, IL-1 receptor-associated protein kinase, and tumor necrosis factor (TNF) receptor-associated factor 6, leading to the activation of nuclear factor (NF)-{kappa}B [10 ]. The fact that the mouse TERT promoter is regulated by the widely expressed and highly inducible NF-{kappa}B transcription factor further supports our findings of telomerase activation in DC maturation [29 ]. However, the physiological role of the inducible telomerase activity is still unclear. In addition to the well-characterized action of telomerase in adding telomeric repeats, it should be noted that recent studies suggest that telomerase may have additional functions, for example, in protecting cellular, proliferative capacity and in preventing apoptosis without telomere elongation [30 ]. Actually in our experiments, during differentiation to mDCs from imDCs by microbial components, telomerase activity was increased without cellular proliferation (data not shown). Engagement of TLRs, CD40L, or TNF-related activation-induced cytokine receptor enhances longevity of DCs through NF-{kappa}B activation [31 , 32 ], which also positively controls telomerase expression. Considering recent findings that LPS and CpG not only mature DCs but also inhibit DC apoptosis [33 , 34 ], telomerase activation may contribute to this antiapoptotic effect. In addition, DCs were reported to be targets of cytotoxic T cell and NK cell-mediated killing, and activation of DCs by LPS or CD40 renders DCs resistant to the cytotoxicity [35 , 36 ]. Taken together, the inducible telomerase activity could be a key component to potentiate DC function, proliferation, and survival.

DCs in aged mice
Despite numerous, recent advances in the molecular and cellular biology of DCs, very few groups have addressed the topic of DCs and aging [37 ]. In animal models of old mice, DCs of lymph nodes showed degenerative characteristics with decreased adhesion molecule expression, less dendrite formation, and reduced antigen-trapping capacity, which together, imply disruption of functional activity [38 ]. In contrast, DCs generated from peripheral blood of elderly people were not impaired in their capacity to induce cell responses [39 ]. During aging, however, it is obvious that there is a marked decline in the reactivity of the immune system, which has been attributed to impairment of lymphocyte function in corporation with innate-immune responses [40 , 41 ]. Here, we observed that splenic DCs in old mice possessed much higher telomerase activity than young mice but even decreased the activity after LPS stimulation (Fig. 3A) . On the contrary, BM DCs from old mice had considerably lower telomerase activity than those from younger ones and remained weaker after stimulation with microbial components, together with lower activation marker expression (Fig. 4) . Thus, splenic DCs and BM DCs have the different nature in old mice. The mechanisms and physiological meanings underlying this contradicting data between splenic and BM DCs are still to be investigated. As lower telomerase activity of BM DCs in old mice seemed associated with an insufficiently activated status (Fig. 4C) , it may reflect inferior differentiation and regeneration potential of BM cells. Conversely, peripheral splenic DCs in old mice may be compensating for something such as telomere shortening or functional defects of DCs and implying a terminal stage in cell fate. However, poor responses of expression of activation markers to LPS stimulation were consistent in splenic and BM DCs (Figs. 3B and 4C) . In any case, DCs in old mice have a different feature from younger mice, and these may contribute to the pathogenesis and poor resistance to infection or endotoxin shock accompanied with aging

This is the first report to investigate the telomerase activity in DCs and make a comparison between young and old mice. Our observations would provide a new insight regarding immunobiology and immunosenescense. Analysis of DCs in telomerase knockout mice (mTERT-/-) [42 ] will clarify the physiological roles of telomerase in immunity.


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ACKNOWLEDGEMENTS
 
The study was supported by the Fund for Comprehensive Research on Aging and Health, the Research Grant for Longevity Sciences (13C-1) from the Ministry of Health and Welfare, the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, a grant from Japan Health Science Foundation, and grant-in-aids for Scientific Research from the Ministry of Education and Uehara Memorial Foundation. We thank Dr. Kerry S. Campbell and Akiko Maki (Fox Chase Cancer Center, Philadelphia, PA) for critically reading the manuscript and helpful discussion.


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FOOTNOTES
 
Current address of Lin Ping: Institute of Cancer Research, West China Hospital, Sichuan University, China.

Received January 10, 2003; accepted April 2, 2003.


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